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EP2298359A1 - Nucleic acid capable of controlling degranulation of mast cell - Google Patents

Nucleic acid capable of controlling degranulation of mast cell Download PDF

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Publication number
EP2298359A1
EP2298359A1 EP09758404A EP09758404A EP2298359A1 EP 2298359 A1 EP2298359 A1 EP 2298359A1 EP 09758404 A EP09758404 A EP 09758404A EP 09758404 A EP09758404 A EP 09758404A EP 2298359 A1 EP2298359 A1 EP 2298359A1
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EP
European Patent Office
Prior art keywords
mir
hsa
nucleic acid
nucleotide sequence
mast cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09758404A
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German (de)
French (fr)
Inventor
Kyoko Kosaka
Yoji Yamada
Kasumi Miura
Tatsuya Miyazawa
Tetsuo Yoshida
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Kyowa Kirin Co Ltd
Original Assignee
Kyowa Hakko Kirin Co Ltd
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Publication of EP2298359A1 publication Critical patent/EP2298359A1/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/713Double-stranded nucleic acids or oligonucleotides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • A61P11/06Antiasthmatics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/08Antiallergic agents
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/14Type of nucleic acid interfering N.A.
    • C12N2310/141MicroRNAs, miRNAs
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00Production
    • C12N2330/10Production naturally occurring

Definitions

  • the present invention relates to a mast cell degranulation control agent, a diagnostic agent or therapeutic agent for a disease resulting from an abnormality of mast cell degranulation, control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent.
  • Micro-RNA which is one kind of nucleic acids, is a small non-coding single-stranded RNA of about 22 nucleotides that is not translated into a protein, and has known as being present in many types in organisms, including humans (non-patent documents 1 and 2).
  • a micro-RNA is produced from a gene transcribed to a single or clustered micro-RNA precursor.
  • a primary-miRNA which is a primary transcript
  • a precursor-miRNA (pre-miRNA) of about 70 nucleotides having a characteristic hairpin structure is produced from the pri-miRNA.
  • the mature type micro-RNA is produced from the pre-miRNA by Dicer-mediated processing (non-patent document 3).
  • a mature type micro-RNA is thought to be involved in the post-transcriptional control of gene expression by complementarily binding to an mRNA for a target to suppress the translation of the mRNA, or to degrade the mRNA.
  • a micro-RNA suppresses the expression of the mRNA for a target by binding to a partially complementary sequence in the 3'-untranslational region (3'-UTR) of the target mRNA to suppress the translation thereof, or to degrade the target mRNA.
  • the complementarity of the 2nd to 8th nucleotides from the 5'-end of the micro-RNA is particularly important; this region is sometimes called the "seed sequence" of the micro-RNA (non-patent document 4). It has been shown that micro-RNAs having a same seed sequence suppress the expression of a same target mRNA even if their other sequences differ. Therefore, by making a sequence complementary to a sequence present at the 3'-end of an optionally chosen mRNA as the seed sequence, an RNA having micro-RNA-like activity can be designed.
  • the term micro-RNA unlike siRNA, often refers exclusively to an RNA that occurs naturally in cells, so a micro-RNA-like sequence designed as described above is sometimes particularly referred to as an artificial micro-RNA.
  • micro-RNA database miRBase (http://microrna.sanger.ac.uk/) 533 species of micro-RNAs were registered for humans, and 5,071 species for all organisms.
  • miR-181 which is involved in hematopoietic lineage differentiation
  • miR-375 which is involved in insulin secretion
  • Non-patent Documents 8 to 10 These mediators are diverse and include amines, arachidonic acid metabolites, proteases, cytokines, chemokines and the like.
  • mast cells are thought to play major roles in the pathogenesis of various allergic diseases; therefore, it is thought that by controlling a function of mast cells, treatment of allergic diseases is possible.
  • existing therapeutic agents for allergies are known to possess the action of suppressing the release of inflammatory mediators from mast cells
  • their studies have traditionally been conducted mainly using rodent mast cells, and there have been no adequate investigations of whether the existing therapeutic agents are actually effective on human mast cells.
  • rodent mast cells and human mast cells have different reactivities to drugs (non-patent document 9).
  • sodium cromoglicate which was used as a suppressant of inflammatory mediator release, remarkably suppressed the IgE-dependent release of inflammatory mediators in rat abdominal mast cells, but the action thereof on human mast cells was not potent (non-patent document 12).
  • Azelastine hydrochloride at high concentrations, suppressed the release of histamine, PGD2, and LT and production of GM-CSF and MIP-1 ⁇ , from human mast cells in culture, but none of these activities were potent.
  • Suplatast tosilate which was used as an anti-cytokine drug, exhibited inflammatory mediator release suppressive action on rat mast cells, but lacked action on human mast cells (non-patent document 12).
  • Non-patent Document 13 the ⁇ 2 adrenaline receptor stimulant isoproterenol suppresses the release of histamine, LT, PGD2, GM-CSF and MIP-1 ⁇ from cultured human mast cells by 80% or more at a low concentration of 10 nmol/l.
  • Non-patent Document 15 an analysis of micro-RNAs expressed in mouse marrow-derived mast cells (Non-patent Document 15) is the only available relevant report, with no reports on micro-RNAs expressed in human mast cells. Taking into account the interspecific differences between humans and mice, it is difficult to predict information on the expression of micro-RNAs in human mast cells on the basis of information on the expression of micro-RNAs in mouse mast cells. Additionally, there is no knowledge about the involvement of micro-RNAs in the diverse functions of mast cells.
  • nucleic acids such as micro-RNAs and precursors thereof expressed in various human organs, and analyzing the functions thereof to elucidate their relations to diseases, new therapeutic agents and diagnostic agents will be developed.
  • finding nucleic acids, such as micro-RNAs and precursors thereof, that act in mast cells is expected to lead to the elucidation of functions such as mast cell differentiation and degranulation, cytokine-producing mast cell differentiation and degranulation, inflammatory mediator production, cytokine production, and chemokine production, and hence lead to the development of methods of mast cell isolation, cultivation, differentiation control, degranulation control, inflammatory mediator production control, cytokine production control, and chemokine production control, and new therapies for allergic diseases and the like involving the utilization thereof.
  • the present invention relates to the following (1) to (18).
  • a mast cell degranulation control agent a diagnostic agent or therapeutic agent for a disease resulting from mast cell degranulation control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent, all of which involve the use of a nucleic acid and the like.
  • the nucleic acid used in the present invention may be any molecule, as far as it is a molecule resulting from polymerization of a nucleotide or a molecule functionally equivalent to the nucleotide; for example, an RNA, which is a ribonucleotide polymer, a DNA, which is a deoxyribonucleotide polymer, a mixed polymer of RNA and DNA, and a nucleotide polymer, including a nucleotide analogue, can be mentioned; furthermore, the nucleic acid may be a nucleotide polymer, including a nucleic acid derivative.
  • nucleic acid in the present invention may be a single-stranded nucleic acid or a double-stranded nucleic acid.
  • Double-stranded nucleic acids include double-stranded nucleic acids wherein one strand hybridizes with the other strand under stringent conditions.
  • the nucleotide analogue may be any molecule, as far as it is a molecule prepared by modifying a ribonucleotide, a deoxyribonucleotide, an RNA or a DNA in order to improve the nuclease resistance thereof, to stabilize the same, to increase the affinity thereof for a complementary chain nucleic acid, to increase the cell permeability thereof, or to visualize the same, compared with the RNA or DNA; for example, a nucleotide analogue modified at the sugar moiety thereof, a nucleotide analogue modified at phosphoric acid diester bond and the like can be mentioned.
  • the nucleotide analogue modified at the sugar moiety thereof may be any one, as far as an optionally chosen chemical structural substance has been added to, or substituted for, a portion or all of the chemical structure of the sugar of the nucleotide; for example, a nucleotide analogue substituted by 2'-O-methylribose, a nucleotide analogue substituted by 2'-O-propylribose, a nucleotide analogue substituted by 2'-methoxyethoxyribose, a nucleotide analogue substituted by 2'-O-methoxyethylribose, a nucleotide analogue substituted by 2'-O-[2-(guanidium)ethyl]ribose, a nucleotide analogue substituted by 2'-O-fluororibose, a bridged nucleic acid (BNA) having two cyclic structures as a result
  • the nucleotide analogue modified at phosphoric acid diester bond may be any one, as far as an optionally chosen chemical substance has been added to, or substituted for, a portion or all of the chemical structure of the phosphoric acid diester bond of the nucleotide; for example, a nucleotide analogue substituted by a phosphorothioate bond, a nucleotide analogue substituted by an N3'-P5' phosphoamidate bond, and the like can be mentioned [ SAIBO KOGAKU, 16, 1463-1473 (1997 )] [RNAi Method and Antisense Method, Kodansha (2005)].
  • the nucleic acid derivative may be any molecule, as long as it is a molecule prepared by adding another chemical substance to the nucleic acid in order to improve the nuclease resistance thereof, to stabilize the same, to increase the affinity thereof for a complementary chain nucleic acid, to increase the cell permeability thereof, or to visualize the same, compared with the nucleic acid; for example, a 5'-polyamine conjugated derivative, a cholesterol conjugated derivative, a steroid conjugated derivative, a bile acid conjugated derivative, a vitamin conjugated derivative, a Cy5 conjugated derivative, a Cy3 conjugated derivative, a 6-FAM conjugated derivative, a biotin conjugated derivative and the like can be mentioned.
  • nucleic acids used in the present invention the following nucleic acids (a) to (k) can be mentioned.
  • micro-RNAs are preferably used.
  • a micro-RNA refers to a single-stranded RNA 17 to 28 nucleotides long.
  • the peripheral genome sequence, including the sequence, of a micro-RNA has a sequence capable of forming a hairpin structure, and the micro-RNA can be cut out from either one strand of the hairpin.
  • a micro-RNA complementarily binds to an mRNA serving as a target therefor to suppress the translation of the mRNA or promote the decomposition of the mRNA, thereby mediating the post-translational control of gene expression.
  • micro-RNAs used in the present invention human micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs: 1 to 150 and 3372 to 3406 can be mentioned. Furthermore, as micro-RNAs having the same function as the function of human micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406, nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs: 151 to 1237 and 3407 to 3684, which are orthologues of the human micro-RNAs, can be mentioned.
  • orthologues of the human micro-RNA of SEQ ID NO:1 those consisting of a nucleotide sequence of any one of SEQ ID NOs:151 to 173 and 3407 can be mentioned.
  • Tables of the correspondence of micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406 and orthologues thereof are shown in Table 1-1 to Table 1-8.
  • the biological species shown in the uppermost fields of Table 1-1 to Table 1-8 are as follows: hsa, Homo sapiens, human being; mmu, Mus musculus, mouse; rno, Rattus norvegicus, rat; xla, Xenopus laevis, African clawed toad; xtr, Xenopus tropicalis, tropical clawed toad; gga, Gallus gallus, chicken; cfa, Canis familiaris, dog; mdo, Monodelphis domestica, gray short-tailed opossum; age, Ateles geoffroyi, black-handed spider monkey; 11a, Lagothrix lagotricha, Humboldt's wooly monkey; sla, Saguinus labiatus, red-bellied tamarin; mml, Macaca mulatta, rhesus monkey; mne, Macaca nemestrina, pig-tailed macaque;
  • micro-RNA suppresses the translation of the mRNA of a target gene therefor
  • an mRNA having a nucleotide sequence complementary to the 2nd to 8th nucleotides from the 5'-end of the micro-RNA is recognized as a micro-RNA target gene ( Current Biology, 15, R458-R460 (2005 )).
  • the expression of the mRNA is suppressed by the micro-RNA. Therefore, micro-RNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5'-end suppress the expression of the mRNA of the same target gene to exhibit the same function.
  • micro-RNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5'-end as the nucleotide sequence of a micro-RNA consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406 nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs:1238 to 1543 and 3685 to 3741 can be mentioned.
  • micro-RNAs for the micro-RNA of SEQ ID ND:1, micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs:1238 to 1286 can be mentioned.
  • Tables of the correspondence of micro-RNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5'-end as the nucleotide sequence of a micro-RNA consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406 are shown in Table 2-1 to Table 2-8. Because micro-RNAs having a common seed sequence are considered to share the same target nucleotide sequence, they are considered to possess similar functions.
  • micro-RNA precursors are also used preferably.
  • a micro-RNA precursor is a nucleic acid about 50 to about 200 nucleotides long, more preferably about 70 to about 100 nucleotides long, including the above-described nucleic acids used in the present invention, and capable of forming a hairpin structure.
  • a micro-RNA is produced from a micro-RNA precursor via processing by a protein called Dicer.
  • micro-RNA precursors used in the present invention for the human micro-RNAs of SEQ ID NO:1 nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 1545 can be mentioned.
  • nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs:1546 to 3371 and 3742 to 4147 can be mentioned.
  • Tables of the correspondence of micro-RNAs and micro-RNA precursors used in the present invention are shown in Table 3-1 to Table 3-39.
  • sequence number miRNA name sequence number miRNA precursor name 151 mmu-miR-16 1709 mmu-mir-16-1 1710 mmu-mir-16-2 152 mo-miR-16 1711 mo-mir-16 153 age-miR-16 1712 age-mir-16 154 lla-miR-16 1713 lla-mir-16 155 sla-miR-16 1714 sla-mir-16 156 mml-miR-16 1715 mml-mir-16-1 1716 mml-mir-16-2 157 mne-miR-16 1717 mne-mir-16 158 ggo-miR-16 1718 ggo-mir-16 159 ppa-miR-16 1719 ppa-mir-16 360 ptr-miR-16 1720 ptr-mir-16 161 ppy-miR-16 1721 ppy-mir-16 162 lca-miR-16 1722 lca-mir-16 163 cfa-miR
  • sequence number miRNA name sequence number miRNA precursor name 201 mml-miR-18 1764 mml-mir-18 202 mne-miR-18 1765 mne-mir-18 203 ggo-miR-18 1766 ggo-mir-18 204 ppa-miR-18 1767 ppa-mir-18 205 ptr-miR-18 1768 ptr-mir-18 206 ppy-miR-18 1769 ppy-mir-18 207 lca-miR-18 1770 lca-mir-18 208 mdo-miR-18 1771 mdo-mir-18 209 gga-miR-18a 1772 gga-mir-18a 210 xtr-miR-18a 1773 xtr-mir-18a 211 bta-miR-18a 1774 bta-mir-18a 212 ssc-miR-18 1775 ssc-mir-18 213 dre-m
  • sequence number miRNA name sequence number miRNA precursor name 251 mo-miR-21 1814 mo-mir-21 252 cgr-miR-21 1815 cgr-mir-21 253 age-miR-21 1816 aga-mir-21 254 mml-miR-21 1817 mml-mir-21 255 mne-miR-21 1818 mne-mir-21 256 Mo-miR-21 1819 ggo-mir-21 257 ppa-miR-21 1820 ppa-mir-21 258 ptr-miR-21 1821 ptr-mir-21 259 ppy-miR-21 1822 ppy-mir-21 260 cfa-miR-21 1823 cfa-mir-21 261 mdo-miR-21 1824 mdo-mir-21 262 gga-miR-21 1825 gga-mir-21 263 bta-miR-21 1826 bta-mir-21 264 s
  • sequence number miRNA name sequence number miRNA precursor name 451 mml-miR-208b 2042 mml-mir-208b 452 mdo-miR-208 2043 mdo-mir-208 453 xtr-miR-208 2044 xtr-mir-208 454 mmu-miR-210 2045 mmu-mir-210 455 mmu-miR-210 2046 mo-mir-210 456 mml-miR-210 2047 mml-mir-210 457 xtr-miR-210 2048 xtr-mir-210 458 dre-miR-210 2049 dre-mir-210 459 fru-miR-210 2050 fru-mir-210 460 tni-miR-210 2051 tni-mir-210 461 mnu-miR-21 2052 mmu-mir-211 462 mo-niR-211 2053 mo-
  • sequence miRNA name sequence miRNA precusor name 551 ptr-miR-194 2165 ptr-mir-194 552 ppy-miR-194 2166 ppy-mir-194 553 cfa-miR-194 2167 cfa-mir-194 554 gga-miR-194 2168 gga-mir-194 555 xtr-miR-194 2169 xtr-mir-194-1 2170 xtr-mir-194-2 556 dre-miR-194b 2171 dre-mir-194b 557 fru-miR-194 2172 fru-mir-194 558 tni-miR-194 2173 tri-mir-194 559 cfa-miR-500 2174 cfa-mir-500 560 mmu-miR-197 2175 mmu--mir-197 561 age-miR-197 2176 age-mir-197 562 mm
  • sequence number miRMA name sequence number miRNA precursor name 601 mml-let-7f 2224 mm-let-7f-1 2225 mml-let-7f-2 602 mml-lest-7g 2226 mml-let-7g 603 mm-let-7i 2227 mml-let-7i 604 cfa-let-7a 2228 cfa-let-7a 605 cfa-let-7c 2229 cfa-let-7c 606 cfa-let-7e 2230 cfa-let-7e 607 cfa-let-7f 2231 cfa-let-7f 608 cfa-let-7g 2232 cfa-let-7g 609 cfa-let-7j 2233 cfa-let-7j 610 mdo-let-7a 2234 mdo-let-7a-1 2235 mdo-let-7a-2 2236 mdo-let-7a-3 611 mdo-let-7b 22
  • sequence number miRNA name sequence number miRNA precursor name 701 gga-miR-9 2362 gga-mir-9-1 2363 gga-mir-9-2 702 xtr-miR-9 2364 xtr-mir-9-3 703 xtr-miR-9b 2365 xtr-mir-3b 704 dre-miR-9 2366 dre-mir-9-1 2367 dre-mir-9-2 2368 dre-mir-9-3 2369 dre-mir-9-4 2370 dre-mir-9-5 2371 dre-mir-9-6 2372 dre-mir-9-7 705 fru-miR-9 2373 fru-mir-9-1 2374 fru-mir-9-2 2375 fru-mir-9-3 2376 fru-mir-9-4 706 tni-miR-9 2377 tni-mir-9-1 2378 tn-mir-9-2 2379 tni-mir-9-3 2380 tni-mir-9-4 707 mm
  • sequence number miRNA name sequence number miRNA precursor name 851 ppy-miR-106a 2534 ppy-mir-106a 852 ppy-miR-106b 2535 ppy-mir-106b 853 cfa-miR-106b 2536 cfa-mir-106b 854 gga-miR-106 2537 gga-mir-106 855 xtr-miR-106 2538 xtr-mir-106 856 bta-miR-106 2539 bta-mir-106 857 ssc-miR-106a 2540 ssc-mir-106a 858 rno-miR-126* 2541 rno-mir-126 859 gga-miR-126* 2542 gga-mir-126 860 xtr-miR-126* 2543 xtr-mir-126 861 bta-miR-126* 2544 bta-
  • sequence number miRNA name sequence number miRNA precursor name 901 mne-miR-133a 2597 mne-mir-133a 902 ggo-miR-133a 2598 ggo-mir-133a 903 ppa-miR-133a 2599 ppa-mir-133a 904 ptr-miR-133a 2600 ptr-mir-133a 905 mdo-miR-133a 2601 mdo-mir-133a 906 gga-miR-133a 2602 gga-mir-133a-1 2603 gga-mir-133a-2 907 gga-miR-133c 2604 gga-mir-133c 908 xla-miR-133a 2605 xla-mir-133a 909 xtr-miR-133a 2606 xtr-mir-133a 910 xtr-miR-133d 2607 xtr-mir
  • sequence number miRNA name sequence number miRNA precusor name 951 fru-miR-142a 2659 fru-mir-142a 952 fru-miR-142b 2660 fru-mir-142b 953 tni-miR-142a 2661 tni-mir-142a 954 tni-miR-142b 2662 tni-mir-142b 955 mmu-miR-146a 2663 mmu-mir-146a 956 rno-miR-146a 2664 rno-mir-146a 957 mml-miR-146a 2665 mml-mir-146a 958 cfa-miR-146a 2666 cfa-mir-146a 959 gga-miR-146a 2667 gga-mir-146a 960 dre-miR-146a 2668 dre-mir-146a 961 mmu-miR-146b
  • sequence number miRNA name sequence number miRNA precusor name 1001 gga-miR-181a 2721 gga-mir-181a-1 2722 gga-mir-181a-2 1002 gga-miR-181b 2723 gga-mir-181b-1 2724 gga-mir-181b-2 1003 xtr-miR-181a 2725 xtr-mir-181a-1 2726 xtr-mir-181a-2 1004 xtr-miR-181b 2727 xtr-mir-181b-1 2728 xtr-mir-181b-2 1005 bta-miR-181a 2729 bta-mir-181a 1006 bta-miR-181 b 2730 bta-mir-181b 1007 bta-miR-181c 2731 bta-mir-181c 1008 ssc-miR-181b 2732 ssc-mir
  • sequence number miRNA name sequence number miRNA precursor name 1351 mdo-miR-103 3112 mdo-mir-103-1 3113 mdo-mir-103-2 1352 gga-miR-103 3114 gga-mir-103-1 3115 gga-mir-103-2 1353 xtr-miR-103 3116 xtr-mir-103-1 3117 xtr-mir-103-2 1354 bta-miR-103 3118 bta-mir-103-1 3119 bta-mir-103-2 1355 ssc-miR-103 3120 ssc-mir-103 1356 dre-miR-103 3121 dre-mir-103 1357 fru-miR-103 3122 fru-mir-103 1358 tni-miR-103 3123 tni-mir-103 1359 hsa-miR-148a 3124 hsa-mir-148a 1360 h
  • sequence number miRNA name sequence number miRNA precursor name 1401 hsa-miR-519e* 3167 hsa-mir-519e 1402 hsa-miR-302a 3168 hsa-mir-302a 1403 hsa-miR-302b 1665 hsa-mir-302b 1404 hsa-miR-302c 1666 hsa-mir-302c 1405 hsa-miR-302d 3169 hsa-mir-302d 1406 hsa-miR-302e 3170 hsa-mir-302e 1407 hsa-miR-372 3171 hsa-mir-372 1408 hsa-miR-373 1668 hsa-mir-373 1409 hsa-miR-520a-3p 3172 hsa-mir-520a 1410 hsa-miR-520b 3173 hsa-mir-520b
  • sequence number miRNA name sequence number miRNA precursor name 1472 age-miR-98 3301 age-mir-98 1473 mml-miR-98 3302 mml-mir-98 1474 ggo-miR-98 3303 ggo-mir-98 1475 ppa-miR-98 3304 ppa-mir-98 1476 ptr-miR-98 3305 ptr-mir-98 1477 ppy-miR-98 3306 ppy-mir-98 1478 cfa-miR-98 3307 cfa-mir-98 1479 xtr-miR-98 3308 xtr-mir-98 1480 bta-miR-98 3309 bta-mir-98 1481 hsa-miR-23a 3310 hsa-mir-23a 1482 hsa-miR-130a* 3311 hsa-mir-130a 1483 hsa-m
  • sequence number miRNA name sequence number miRNA precursor name 3455 xtr-miR-9a 3832 xtr-mir-9a-1 3833 xtr-mir-9a-2 3456 ssc-miR-9-1 3834 ssc-mir-9-1 3835 ssc-mir-9-2 3457 ssc-miR-9-2 3834 ssc-mir-9-1 3835 ssc-mir-9-2 3458 mmu-miR-23a 3836 mmu-mir-23a 3459 mmu-miR-27a 3837 mmu-mir-27a 3460 xtr-miR-31b 3838 xtr-mir-31b 3461 mmu-miR-33 3839 mmu-mir-33 3462 mmu-miR-106b 3840 mmu-mir-106b 3463 mmu-miR-126-5p 3841 mmu-mir-126 3464 cfa-mi
  • sequence number miRHA name sequence number miRNA precursor name 3503 mo-miR-29b 3881 mo-mir-29b-1 3882 mo-mir-29b-2 3504 mo-miR-29c 3883 mo-mir-29c 3505 age-miR-29b 3884 age-mir-29b 3506 Ila-miR-29b 3885 Ila-mir-29b 3507 sla-miR-29b 3886 sla-mir-29b 3508 mml-miR-29b 3887 mml-mir-29b-1 3888 mml-mir-29b-2 3509 mml-miR-29c 3889 mml-mir-29c 3510 mne-miR-29b 3890 mne-mir-29b 3511 ggo-miR-29b 3891 ggo-mir-29b-1 3892 ggo-mir-29b-2 3512 ppa-miR-29b 3893 ppa-
  • sequence number miRRA name sequence number miRNA precursor name 3593 dre-miR-30d 3980 dre-mir-30d 3594 dre-miR-30e 1929 dre-mir-30e-2 3595 fru-miR-30b 3981 fru-mir-30b 3596 fru-miR-30c 3982 fru-mir-30c 3597 fru-miR-30d 3983 fru-mir-30d 3598 tni-miR-30b 3984 tni-mir-30b 3599 tni-miR-30c 3985 tni-mir-30c 3600 tni-miR-30d 3986 tni-mir-30d 3601 lla-miR-105 3987 lla-mir-105 3602 sla-miR-105 3988 sla-mir-105 3603 mml-miR-105 3989 mml-mir-105-1 3990 mml-mir-105-2 3604 mne-m
  • sequence number miRNA name sequence number miRNA precursor name 3644 mml-miR-383 4033 mml-mir-383 3645 cfa-miR-383 4034 cfa-mir-383 3646 mdo-miR-383 4035 mdo-mir-383 3647 gga-miR-383 4036 gga-mir-383 3648 xb-miR-383 4037 xtr-mir-383 3649 mmu-miR-411 4038 mmu-mir-411 3650 mo-miR-411 4039 mo-mir-411 3651 mml-miR-411 4040 mml-mir-411 3652 mmu-miR-423-3p 4041 mmu-mir-423 3653 mo-miR-423 4042 mo-mir-423 3654 mml-miR-423-3p 4043 mml-mir-423 3655 mmu-miR-433 4044 mmu-mir
  • sequence number mRNAname sequence number miRNA precursor name 3701 mo-miR-130a 4089 mo-mir-130a 3702 mo-miR-130b 4090 mo-mir-130b 3703 mo-miR-301 a 4091 mo-mir-301a 3704 mo-miR-301b 4092 mo-mir-301b 3705 mml-miR-130a 4093 mml-mir-130a 3706 mml-miR-130b 4094 mml-mir-130b 3707 mml-miR-301a 4095 mml-mir-301a 3708 mml-miR-301b 4096 mml-mir-301b 3709 mme-miR-130a 4097 mne-mir-130a 3710 ggo-miR-130a 4098 ggo-mir-130a 3711 ppa-miR-130a 4099 ppa-mir-130a 3712 cfa-miR
  • a nucleic acid having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1 to 4147 means a nucleic acid having an identity of at least 90% or more, preferably 93% or more, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, most preferably 98% or more, to a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 3371, as calculated using an analytical software program such as BLAST ( J. Mol. Biol., 215, 403 (1990 )) or FASTA ( Methods in Enzymology, 183, 63 (1990 )).
  • BLAST J. Mol. Biol., 215, 403 (1990 )
  • FASTA Methods in Enzymology, 183, 63 (1990 )
  • stringent conditions are conditions that allow signals to be detected by adding to a membrane blotted with one strand the other strand labeled with 32 P-ATP in a hybridization buffer consisting of 7.5 mL, 0.6 mL of 1M Na 2 HPO 4 (pH 7.2), 21 mL of 10% SDS, 0.6 mL of 50xDenhardt's solution, and 0.3 mL of 10 mg/mL sonicated salmon sperm DNA, carrying out a reaction at 50°C overnight, thereafter washing the membrane with 5xSSC/5% SDS liquid at 50°C for 10 minutes, and further washing the same with 1xSSC/1% SDS liquid at 50°C for 10 minutes, thereafter taking out the membrane, and applying it to an X-ray film.
  • a hybridization buffer consisting of 7.5 mL, 0.6 mL of 1M Na 2 HPO 4 (pH 7.2), 21 mL of 10% SDS, 0.6 mL of 50xDenhardt's solution, and 0.3
  • the method of detecting the expression of nucleic acid such as a micro-RNA using a nucleic acid may be any method, as far as the presence of nucleic acid such as a micro-RNA or micro-RNA precursor in a sample can be detected; for example, (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like can be mentioned.
  • the method of detecting the amount expressed of a nucleic acid such as a micro-RNA, a mutation of the nucleic acid, or a mutation of the genome that encodes the nucleic acid using a nucleic acid used in the present invention may be any method, as far as it enables detection of a mutation of the nucleotide sequence of a nucleic acid such as a micro-RNA or micro-RNA precursor in a sample; for example, a method comprising detecting a hetero-double-strand formed by hybridization of a nucleic acid having a non-mutated nucleotide sequence and a nucleic acid having a mutated nucleotide sequence, a method comprising directly sequencing a sample-derived nucleotide sequence to detect the presence or absence of a mutation, and the like can be mentioned.
  • the vector that expresses a nucleic acid used in the present invention may be any vector, as far as it has been designed to biosynthesize a nucleic acid, such as a micro-RNA, used in the present invention, when introduced to, and transcribed in, a cell.
  • pcDNA6.2-GW/miR manufactured by Invitrogen Company
  • pSilencer 4.1-CMV manufactured by Ambion Company
  • pSINsi-hH1 DNA manufactured by Takara Bio Company
  • pSINsi-hU6 DNA manufactured by Takara Bio Company
  • pENTR/U6 manufactured by Invitrogen Company
  • the method of suppressing the expression of a gene having a target nucleotide sequence for a nucleic acid, such as a micro-RNA, used in the present invention may be any method, as far as it suppresses the expression of a gene having the target nucleotide sequence.
  • a target gene may be any method, as far as it suppresses the expression of a gene having the target nucleotide sequence.
  • to suppress the expression encompasses a case where the translation of an mRNA is suppressed, and a case where cleavage or degradation of an mRNA results in a decreased amount of protein translated from the mRNA.
  • substances that suppress the expression of an mRNA having the target nucleotide sequence specifically nucleic acids such as siRNAs and antisense oligonucleotides can be mentioned.
  • the siRNAs can be prepared on the basis of information on the continuous sequence of the mRNA ( Genes Dev., 13, 3191 (1999 )).
  • the number of nucleotide residues constituting one strand of the siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.
  • Micro-RNAs used in the present invention also include an artificial micro-RNA that is a single-stranded RNA 17 to 28 nucleotides long, comprising a sequence complementary to a 7-nucleotide continuous sequence present in a gene (target gene) having a target nucleotide sequence for a micro-RNA of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741 as the 2nd to 8th nucleotides.
  • target gene a target nucleotide sequence for a micro-RNA of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741 as the 2nd to 8th nucleotides.
  • the extended sequence is called an artificial micro-RNA precursor.
  • a target nucleotide sequence for a micro-RNA refers to the nucleotide sequence of a nucleic acid consisting of several nucleotides recognized by a micro-RNA used in the present invention, wherein the expression of the mRNA having the nucleotide sequence is suppressed by the micro-RNA.
  • a nucleotide sequence complementary to the 2nd to 8th nucleotides from the 5-end of a micro-RNA used in the present invention can be mentioned as a target nucleotide sequence of the micro-RNA.
  • the target nucleotide sequence can be determined.
  • a set of 3' UTR nucleotide sequences of human mRNAs can be prepared using information on genomic sequences and gene positions that can be acquired from "UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway)".
  • genes having a target nucleotide sequence of a micro-RNA of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741 the genes shown in Table 4-1 to Tables 4 to 148, represented by names (Official Symbols and Gene IDs) used in the EntreGene database (http://www.ncbi.nlm.nih.gov/Entrez/) of the US National Center for Biotechnology Information (NCBI), can be mentioned.
  • the substance that controls the expression or a function of a nucleic acid such as a micro-RNA, used in the present invention, may be any substance such as a low-molecular compound, antibody, or siRNA, as far as it inhibits the expression or a function of the nucleic acid, and an siRNA is particularly preferable.
  • substances that suppress the expression of a nucleic acid such as a micro-RNA, used in the present invention, substances that inhibit the function of a factor that is essential to the biosynthesis of the micro-RNA can also be used.
  • Factors that are essential to the biosynthesis of a micro-RNA include, for example, Dicer, TRBP, Exportin5, Drosha, DGCR8 and the like.
  • a method of expressing a nucleic acid such as a micro-RNA in cells in the present invention a method using, for example, besides a gene encoding mRNA, a nucleic acid that causes the expression of the micro-RNA and the like when introduced into the cells can be mentioned.
  • a nucleic acid a DNA, an RNA, or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used.
  • the nucleic acid can be designed in the same way as with Pre-miR TM miRNA Precursor Molecules (manufactured by Ambion) or miRIDIAN microRNA Mimics (manufactured by GE Healthcare), and the nucleic acid such as the micro-RNA of the present invention can be expressed in cells.
  • any method can be used to express a micro-RNA, as far as it allows the micro-RNA to be finally formed in the cells; for example, (1) a method wherein a single-stranded RNA that is a precursor of the micro-RNA is introduced, as well as (2) a method wherein an RNA consisting of double-strand consisting of the micro-RNA as it is and a complementary strand for the micro-RNA, which completely complement each other, is introduced, and (3) a method wherein a double-stranded RNA assumed to have resulted from cleavage of a micro-RNA with a Dicer is introduced, can be mentioned.
  • miCENTURY OX Precursor manufactured by B-Bridge
  • miCENTURY OX siMature manufactured by B-Bridge
  • miCENTURY OX miNatural manufactured by B-Bridge
  • the method of producing a nucleic acid used in the present invention is not particularly limited; the same can be produced by a method using a publicly known chemical synthesis, or an enzymatic transcription method and the like.
  • methods using a publicly known chemical synthesis the phosphoroamidite method, the phosphorothioate method, the phosphotriester method and the like can be mentioned; for example, the same can be synthesized using the ABI3900 high throughput nucleic acid synthesizer (manufactured by Applied Biosystems).
  • transcription with a plasmid or DNA having a desired nucleotide sequence as the template using a typical phage RNA polymerase for example, T7, T3, or SP6RNA polymerase, can be mentioned.
  • a method of screening a substance that suppresses the expression or a function of a nucleic acid by using the nucleic acid used in the present invention a method can be mentioned wherein a vector that expresses the nucleic acid is introduced into cells, and a substance that promotes or suppresses the expression or a function of an RNA having a target nucleotide sequence therefor is screened for.
  • a pharmaceutical with a nucleic acid used in the present invention as an active ingredient can be used to diagnose or treat a disease caused by an abnormality of mast cell.
  • a mast cell abnormality As diseases caused by a mast cell abnormality, specifically, atopic dermatitis, asthma, chronic obstructive lung disease, and allergic disease and the like can be mentioned. Because abnormalities of mast cells include abnormalities in degranulation, a nucleic acid used in the present invention can be used as a mast cell degranulation control agent, that is, a degranulation promoter or a degranulation suppressant.
  • Mast cell degranulation suppressants are suitably used as prophylactic agents or therapeutic agents for atopic dermatitis, asthma, chronic obstructive pulmonary disease, allergic diseases and the like.
  • Mast cell degranulation promoters are suitably used as immunopotentiators. The present invention is hereinafter described in detail.
  • human mast cells can, for example, be prepared from a human lung, skin, fetal liver and the like by a commonly known method ( J. Immunol. Methods, 169, 153 (1994 ); J. Immunol., 138, 861 (1987 ); J. Allergy Clin. Immunol., 107, 322 (2001 ); J. Immunol. Methods., 240, 101 (2000 )).
  • Human mast cells can also be prepared by culturing mononuclear cells prepared from human umbilical blood, peripheral blood, bone marrow, lung or skin in the presence of a stem cell factor (hereinafter abbreviated SCF) according to a commonly known method ( J. Immunol., 157, 343, (1996 ); Blood, 91, 187 (1998 ); J. Allergy Clin. Immunol., 106, 141 (2000 ); Blood, 97, 1016 (2001 ); Blood, 98, 1127 (2001 ); Blood, 100, 3861 (2002 ); Blood, 97, 2045 (2001 )) to allow the mononuclear cells to differentiate into mast cells.
  • SCF stem cell factor
  • a cell line established from a human mast cell can also be used.
  • human mast cell lines LAD2 ( Leuk. Res., 27, 671 (2003 ); Leuk. Res., 27, 677 (2003 )), which is known to well retain the properties of human mast cells, and the like can be mentioned.
  • the method of extracting total RNA from the mast cells acquired by the various methods described above is not particularly limited, as far as a low-molecular RNA such as a micro-RNA is contained; for example, this extraction can be performed by a method described in Molecular Cloning, 3rd editi on.
  • the total RNA can also be extracted using Trizol (manufactured by Invitrogen Company), ISOGEN (manufactured by Nippon Gene Company), mirVana TM miRNA Isolation Kit (manufactured by Ambion Company) , miRNeasy Mini Kit (manufactured by QIAGEN Company) and the like.
  • a low-molecular RNA can also be cloned from a total RNA containing the low-molecular RNA.
  • a method of cloning a low-molecular RNA specifically, a method wherein separation and cutting out of a low-molecular RNA by 15% polyacrylamide gel electrophoresis as described in Genes & Development 15, 188-200 (2000 ), can be mentioned.
  • a method wherein 5'-adenylation 3'-adapter ligation, 5'-adapter ligation, reverse transcription, PCR amplification, concatemerization, and ligation to a vector are performed sequentially, thereafter the low-molecular RNA is cloned, and the nucleotide sequence of the clone is also determined, as described in Science 294, 858-862 (2001 ).
  • separation and cleavage of low-molecular RNA by 15% polyacrylamide gel electrophoresis, 5' terminal dephosphorylation, 3'-adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, and ligation to a microbead vector are performed sequentially, thereafter the low-molecular RNA is cloned, and the nucleotide sequence of the microbeads is read to determine the nucleotide sequence, whereby nucleotide sequence information of the low-molecular RNA can also be acquired, as described in Nucleic Acids Research 34, 1765-1771
  • Whether or not the low-molecular RNA sequence is a micro-RNA can be determined on the basis of whether or not the criteria described in RNA, 9, 277-279 (2003 ) are met. For example, in cases where the low-molecular RNA was newly acquired and the nucleotide sequence thereof was determined, this can be performed as described below.
  • RNAfold [ Nucleic Acids Research 31, 3429-3431 (2003 )], Mfold [ Nucleic Acids Research 31, 3406-3415 (2003 )] and the like can be used.
  • Existing micro-RNA sequences are registered in a database called miRBase (http://microrna.sanger.ac.uk/); whether or not a micro-RNA is identical to an existing micro-RNA can be determined on the basis of whether or not the sequence thereof is identical to one of the sequences listed therein.
  • nucleic acid such as micro-RNA, a precursor thereof and the like
  • methods by (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like can be mentioned.
  • the Northern hybridization is a method wherein a sample-derived RNA is separated by gel electrophoresis, then transferred to a solid support such as a nylon filter, and an appropriately-labeled probe is prepared on the basis of the nucleotide sequence of the nucleic acid of the present invention, and hybridization and washing are performed, whereby a band specifically bound to the nucleic acid is detected; specifically, for example, this method can be performed as described in Science 294, 853-858 (2001 ) and the like.
  • a labeled probe can be prepared by incorporating a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group and the like in a DNA, RNA, or LNA having a sequence complementary to the nucleotide sequence of the nucleic acid to be used by a method, for example, nick translation, random priming or 5'-terminal phosphorylation. Because the amount of labeled probe bound reflects the expression level of the nucleic acid, the expression level of the nucleic acid can be quantified by quantifying the amount of labeled probe bound. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be achieved by a method described in Molecular Cloning, 3rd editi on.
  • Dot blot hybridization is a method wherein an RNA extracted from a tissue or a cell is spotted in dot forms and immobilized on a membrane, and hybridized with a labeled polynucleotide to be a probe, and an RNA that specifically hybridizes with the probe is detected.
  • the probe used may be the same as that used for Northern hybridization.
  • RNA preparation, RNA spotting, hybridization, and RNA detection can be achieved by a method described in Molecular Cloning, 3rd editi on.
  • In situ hybridization is a method wherein a paraffin-embedded or cryostat-treated section of a tissue acquired from a living organism, or a cell fixed, is used as a sample and subjected to steps for hybridization with a labeled probe and washing, and the distribution and localization of a nucleic acid in the tissue or cell are examined by microscopic examination [ Methods in Enzymology, 254, 419 (1995 )].
  • the probe used may be the same as that used for Northern hybridization. Specifically, a nucleic acid such as micro-RNA etc. can be detected in accordance with a method described in Nature Method 3, 27 (2006 ).
  • a cDNA synthesized from a sample-derived RNA using a primer for reverse transcription and a reverse transcriptase (hereunder, this cDNA is also referred to as a sample-derived cDNA) is used for the measurement.
  • a primer for reverse transcription to be supplied for cDNA synthesis a random primer or a specific RT primer and the like can be used.
  • a specific RT primer refers to a primer having a sequence complementary to a nucleotide sequence corresponding to a nucleic acid and a genomic sequence therearound.
  • a sample-derived cDNA is synthesized, after which a PCR is performed with this cDNA as the template, using a template-specific primer designed from a nucleotide sequence corresponding to a nucleic acid such as micro-RNA or micro-RNA precursor and a genomic sequence therearound, or from a nucleotide sequence corresponding to a primer for reverse transcription, to amplify a cDNA fragment containing the nucleic acid used in the present invention, and the amount of the nucleic acid of the present invention contained in the sample-derived RNA is detected from the number of cycles for reach to a given amount of the fragment.
  • a template-specific primer designed from a nucleotide sequence corresponding to a nucleic acid such as micro-RNA or micro-RNA precursor and a genomic sequence therearound, or from a nucleotide sequence corresponding to a primer for reverse transcription
  • an appropriate region corresponding to a nucleic acid and a genomic sequence therearound is selected, and a pair of a DNA or LNA consisting of a sequence of 20 to 40 nucleotides at the 5' terminus of the nucleotide sequence of the region, and a DNA or LNA consisting of a sequence complementary to a sequence of 20 to 40 nucleotides at the 3' terminus, can be used. Specifically, this can be performed in accordance with a method described in Nucleic Acids Research, 32, e43 (2004 ) and the like.
  • a specific RT primer having a stem-loop structure can also be used as the primer for reverse transcription to be supplied for cDNA synthesis. Specifically, this can be performed using a method described in Nucleic Acid Research, 33, e179 (2005 ), or TaqMan MicroRNA Assays (manufactured by Applied Biosystems). Furthermore, by hybridizing a sample-derived cDNA to a DNA, or LNA corresponding to a nucleotide sequence comprising at least one or more of nucleic acids such as micro-RNA, micro-RNA precursor and the like to be used in the present invention immobilized on a substrate such as a filter, glass slide, or silicone, and performing washing, a change in the amount of the nucleic acid can be detected.
  • a substrate such as a filter, glass slide, or silicone
  • nucleic acids such as micro-RNA and the like can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004 ), Nucleic Acid Research, 32, e188 (2004 ), RNA, 13, 151-159 (2007 ) and the like.
  • a micro-RNA can be detected or quantified in the same manner as mirVana miRNA Bioarray (manufactured by Ambion).
  • a promoter sequence such as the T7 promoter or the SP6 promoter is bound to the 3' terminus of a nucleotide sequence corresponding to the nucleic acid such as micro-RNA or micro-RNA precursor to be used in the present invention or a genomic sequence therearound, and a labeled antisense RNA is synthesized with an in vitro transcription system using a labeled NTP (a mixture of ATP, GTP, CTP, and UTP) and an RNA polymerase.
  • NTP a mixture of ATP, GTP, CTP, and UTP
  • the labeled antisense RNA is bound to a sample-derived RNA to form an RNA-RNA hybrid, after which the hybrid is digested with ribonuclease A, which degrades single-stranded RNAs only.
  • the digest is subjected to gel electrophoresis to detect or quantify an RNA fragment protected against the digestion by forming the RNA-RNA hybrid, as a nucleic acid.
  • the fragment can be detected or quantified using the mirVana miRNA Detection Kit (manufactured by Ambion).
  • the nucleic acid to be used in the present invention such as a micro-RNA or micro-RNA precursor can synthesize not only an RNA, which is a polymer of a ribonucleotide, but also a DNA, which is a polymer of a deoxyribonucleotide, on the basis of the nucleotide sequences.
  • RNA which is a polymer of a ribonucleotide
  • DNA which is a polymer of a deoxyribonucleotide
  • the nucleotide sequence of a DNA can be determined.
  • the nucleotide sequence of a DNA corresponding to the nucleotide sequence of an RNA can be determined, without exception, by reading the U (uracil) contained in the sequence of the RNA as T (thymine).
  • a polymer being a mixture of a ribonucleotide and a deoxyribonucleotide and a polymer comprising a nucleotide analogue and a derivative of a nucleic acid can also be synthesized in the same manner.
  • the method of synthesizing a nucleic acid used in the present invention is not particularly limited; a method using a publicly known chemical synthesis, or an enzymatic transcription method and the like can be mentioned.
  • methods using a publicly known chemical synthesis the phosphoroamidite method, the phosphorothioate method, the phosphotriester method and the like can be mentioned; for example, a nucleic acid can be synthesized using the ABI3900 high throughput nucleic acid synthesizer (manufactured by Applied Biosystems).
  • a method by transcription with a plasmid or DNA having a desired nucleotide sequence as the template using a typical phage RNA polymerase, for example, T7, T3, or SP6RNA polymerase, can be mentioned.
  • a method of detecting a function of a nucleic acid such as a micro-RNA a method can be mentioned wherein the function is determined on the basis of whether or not the translation of the RNA having a target nucleotide sequence is suppressed.
  • Micro-RNAs are known to suppress the translation of an RNA comprising a target nucleotide sequence thereof on the 3' side (3'UTR) [ Current Biology, 15, R458-R460 (2005 )].
  • a DNA wherein a target nucleotide sequence for the single-stranded DNA to be measured is inserted into the 3'UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector, and the expression of the reporter gene is measured when the cell is allowed to express the single-stranded RNA, whereby whether or not a function of a micro-RNA is possessed can be determined.
  • the reporter gene expression vector may be any one, as far as it has a promoter upstream of a reporter gene, and is capable of expressing the reporter gene in the host cell.
  • Any reporter gene can be used; for example, the firefly luciferase gene, the Renilla luciferase gene, the chloramphenicol acetyltransferase gene, the ⁇ -glucuronidase gene, the ⁇ -galactosidase gene, the ⁇ -lactamase gene, the aequorin gene, the green fluorescent protein gene, the DsRed fluorescence gene and the like can be utilized.
  • reporter gene expression vectors having these properties
  • psiCHECK-1 manufactured by Promega
  • psiCHECK-2 manufactured by Promega
  • pGL3-Control manufactured by Promega
  • pGL4 manufactured by Promega
  • pRNAi-GL manufactured by Takara Bio Inc.
  • pCMV-DsRed-Express manufactured by CLONTECH
  • micro-RNA of a single-stranded RNA detection can be achieved as described below.
  • host cells are cultured on a multi-well plate and the like, and a reporter gene expression vector having a target sequence and a single-stranded RNA are expressed. Thereafter, reporter activity is measured both when the single-stranded RNA is and is not expressed, based on which the function of micro-RNA can be detected. 4.
  • Method of detecting a mutation of a nucleic acid such as micro-RNA or micro-RNA precursor As a method of detecting a mutation of a nucleic acid such as a micro-RNA or micro-RNA precursor, a method can be used wherein a heteroduplex formed by hybridization of a normal type nucleic acid and a mutated type nucleic acid are detected.
  • Detection of a heteroduplex by polyacrylamide gel electrophoresis is, for example, performed as described below.
  • a sample-derived DNA or a sample-derived cDNA as a template, and using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid used in the present invention, a fragment smaller than 200 nucleotide pairs is amplified.
  • Heteroduplexs, if formed are slower in mobility than mutation-free homoduplexs, and can be detected as extra bands. In the case of search for a fragment smaller than 200 nucleotide pairs, almost any insertions, deletions, and substitutions of one or more nucleotides can be detected. It is desirable that heteroduplex analysis be performed using a single gel in combination with the single strand conformation analysis described below.
  • a DNA amplified as a fragment smaller than 200 bp with a sample-derived DNA or sample-derived cDNA as the template is denatured, after which it is electrophoresed in non-denatured polyacrylamide gel.
  • the primer By labeling the primer with an isotope or a fluorescent dye at the time of DNA amplification, or by silver-staining the non-labeled amplification product, the amplified DNA can be detected as a band.
  • a control sample may be electrophoresed simultaneously, whereby a fragment with a mutation can be detected on the basis of a difference in mobility.
  • CCM method In chemical cleavage of mismatches (CCM method), a DNA fragment amplified with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid of the present invention is hybridized to a labeled nucleic acid prepared by allowing a nucleic acid to incorporate an isotope or a fluorescent target, and treated with osmium tetraoxide to cleave one strand of the DNA at the mismatched portion, whereby a mutation can be detected.
  • CCM is one of the most sensitive methods of detection, and can be applied to samples of kilobase length.
  • T4 phage resolvase and an enzyme involved in mismatch repair in cells may be used in combination to enzymatically cleave a mismatch.
  • DGGE method denaturing gradient gel electrophoresis
  • a DNA amplified with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid of the present invention is electrophoresed using a gel having a chemical denaturant density gradient or a temperature gradient.
  • the DNA fragment amplified migrates in the gel to a position where it denatures to a single strand, and no longer migrates after the denaturation. Because the migration of the amplified DNA in the gel differs between the presence and absence of a mutation, the presence of the mutation can be detected. To increase the detection sensitivity, the addition of a poly (G:C) end to each primer is effective.
  • a mutation of a nucleic acid used in the present invention can also be detected.
  • Methods of expressing a nucleic acid such as micro-RNA or micro-RNA precursor
  • a nucleic acid used in the present invention can be expressed by using a vector such that a nucleic acid is transcribed and hence biosynthesized when the vector is introduced into a cell. Specifically, by preparing a DNA fragment comprising a hairpin portion on the basis of the aforementioned nucleotide sequence of a nucleic acid or a genomic nucleotide sequence comprising the foregoing nucleotide sequence, and inserting the fragment downstream of a promoter in the expression vector to construct an expression plasmids, and then introducing the expression plasmid into a host cell suitable for the expression vector, whereby the aforementioned nucleic acid can be expressed.
  • an expression vector one capable of self-replication in the host cell, and capable of being incorporated in the chromosome, which comprises a promoter at a position enabling the transcription of a gene comprising the nucleotide sequence of a nucleic acid of the present invention is used.
  • the promoter may be any one, as far as it is capable of expressing in the host cell; for example, a RNA, polymerase II (pol II) system promoter, a RNA polymerase III (pol III) system promoter being a U6RNA and H1RNA transcription system and the like can be mentioned.
  • pol II system promoters the promoter of the cytomegalovirus (human CMV) IE (immediate early) gene, the early promoter of SV40 and the like can be mentioned.
  • expression vectors using them pCDNA6.2-GW/miR (manufactured by Invitrogen) , pSilencer 4.1-CMV (manufactured by Ambion) and the like can be mentioned.
  • pol III system promoters U6RNA, H1RNA or tRNA gene promoters can be mentioned.
  • pSINsi-hH1 DNA manufactured by Takara Bio Inc.
  • pSINsi-hU6 DNA manufactured by Takara Bio Inc.
  • pENTR/U6 manufactured by Invitrogen
  • the gene comprising the nucleic acid used in the present invention can be expressed.
  • the packaging cell may be any cell, as far as it is capable of supplementing a recombinant viral vector deficient in any one of the genes that encode the proteins necessary for the packaging of the virus with the lacked protein; for example, human kidney-derived HEK293 cells, mouse fibroblasts NIH3T3 and the like can be used.
  • proteins derived from a mouse retrovirus such as gag, pol, and env
  • proteins derived from a HIV virus such as gag, pol, env, vpr, vpu, vif, tat, rev, and nef
  • proteins derived from an adenovirus vector such as E1A and E1B
  • proteins such as Rep (p5, p19, p40) and Vp(Cap) can be used.
  • nucleic acid used in the present invention can be introduced directly to the cell without using a vector.
  • nucleic acids useful in this method include not only DNA, RNA or nucleotide analogues, but also chimera molecules thereof, or derivatives of the nucleic acids.
  • a nucleic acid used in the present invention can be expressed.
  • any method can be used, as far as it allows the micro-RNA to be eventually produced in the cell; examples include methods wherein (1) a single-stranded RNA which is a micro-RNA precursor, (2) an RNA, consisting of a double strand consisting of a micro-RNA and a strand complementary to the micro-RNA which are completely complementary to each other, (3) or a double-stranded RNA assuming a state after the micro-RNA is cut by a Dicer, is introduced.
  • miCENTURY OX Precursor manufactured by B-Bridge Company
  • miCENTURY OX siMature manufactured by B-Bridge Company
  • miCENTURY OX miNatural manufactured by B-Bridge Company
  • a nucleic acid such as micro-RNA or micro-RNA precursor to be used in the present invention can be suppressed using an antisense technology ( Bioscience and Industry, 50, 322 (1992 ); Kagaku, 46, 681 (1991 ), Biotechnology, 9, 358 (1992 ), Trends in Biotechnology, 10, 87 (1992 ), Trends in Biotechnology, 10, 152 (1992 ); Cell Technology, 16, 1463 (1997 )], triple helix technology ( Trends in Biotechnology, 10, 132 (1992 )), ribozyme technology ( Current Opinion in Chemical Biology, 3, 274 (1999 ), FEMS Microbiology Reviews, 23, 257 (1999 ), Frontiers in Bioscience, 4, D497 (1999 ), Chemistry & Biology, 6, R33 (1999 ), Nucleic Acids Research, 26, 5237 (1998 ), Trends In Biotechnology, 16, 438 (1998 )), decoy DNA method ( Nippon Rinsho - Japanese Journal of Clinical Medicine, 56, 563 (1998 ), Circulation Research, 82,
  • An antisense refers to one that allows nucleotide sequence-specific hybridization of a nucleic acid having a nucleotide sequence complementary to a certain target nucleic acid to enable the suppression of the expression of the target nucleic acid.
  • a DNA, an RNA or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used. Specifically, by preparing an antisense in accordance with a method described in Nature 432, 226 (2004 ) and the like, the expression can be suppressed.
  • siRNA refers to a short double-stranded RNA comprising the nucleotide sequence of a certain target nucleic acid, that is capable of suppressing the expression of the target nucleic acid by RNA interference (RNAi).
  • RNAi RNA interference
  • the sequence of an siRNA can be designed as appropriate from the target nucleotide sequence on the basis of conditions shown in the literature ( Genes Dev., 13, 3191 (1999 )).
  • an siRNA can be prepared.
  • siRNA expression vector such as pSilencer 1.0-U6 (manufactured by Ambion) or pSUPER (manufactured by OligoEngine)
  • a vector that expresses an siRNA capable of suppressing the expression of the gene can be prepared.
  • any siRNA can be used to suppress a function of a nucleic acid, such as a micro-RNA, used in the present invention, as far as it is capable of suppressing the function of the nucleic acid, preference is given to an siRNA designed on the basis of sequence information on SEQ ID NO:1 to 3371.
  • the number of nucleotide residues constituting one strand of the siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.
  • an antisense or siRNA specific for a nucleic acid such as micro-RNA expressed in mast cells, or a precursor of the micro-RNA
  • the expression of a micro-RNA expressed in mast cells, a precursor of the micro-RNA and the like can be suppressed.
  • an antisense oligonucleotide or siRNA specific for the micro-RNA or the micro-RNA precursor the activation of the micro-RNA can be suppressed, and the action of the micro-RNA or micro-RNA precursor in mast cells can be controlled.
  • an antisense oligonucleotide or siRNA specific for the micro-RNA or precursor thereof in the case of a patient affected by an abnormality of the expression of a micro-RNA expressed in mast cells or a precursor thereof, by administering an antisense oligonucleotide or siRNA specific for the micro-RNA or precursor thereof to the patient, it is possible to control a function of mast cells to treat a disease that develops as a result of the above-described expression abnormality.
  • an antisense oligonucleotide or siRNA that is specific for the micro-RNA or precursor thereof is useful as a therapeutic agent for a disease caused by a cell abnormality of mast cells.
  • an antisense oligonucleotide or siRNA specific for a nucleic acid such as a micro-RNA or a precursor thereof
  • the antisense oligonucleotide or siRNA alone, or a nucleic acid that encodes the same after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, or adeno-associated virus vector
  • an appropriate vector such as a retrovirus vector, adenovirus vector, or adeno-associated virus vector
  • Any method can be used to suppress a function or the expression of the target gene for a nucleic acid used in the present invention, as far as it is based on an activity of a nucleic acid such as a micro-RNA to suppress the expression of the mRNA having the target nucleotide sequence.
  • a method can be mentioned wherein a nucleic acid used in the present invention such as a micro-RNA is expressed to increase the amount of the nucleic acid such as the micro-RNA in cells, whereby the translation of the mRNA having the target sequence is suppressed to suppress the expression of the gene.
  • the expression of a nucleic acid used in the present invention can be achieved by the method described in 5 above.
  • mRNAs having a target nucleotide sequence for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1-1543, and 3372-3741 the respective sets of genes shown in the foregoing Table 4 can be mentioned.
  • siRNA against a target gene shown in Table 4 it is possible to suppress a function of the target gene.
  • a substance that controls, that is, promotes or suppresses the expression or a function of a nucleic acid such as a micro-RNA or a precursor thereof can be screened for using a nucleic acid used in the present invention.
  • a nucleotide sequence to be targeted for screening is chosen from the nucleotide sequence of a micro-RNA or a precursor thereof to be used in the present invention and by means of cells that express a nucleic acid having the nucleotide sequence, a substance that promotes or suppresses the expression or a function of the chosen micro-RNA or precursor thereof can be screened for.
  • cells that express a nucleic acid having the nucleotide sequence of a micro-RNA or micro-RNA precursor, used for screening, mast cells, as well as transformant cells obtained by introducing a vector that expresses a nucleic acid having the nucleotide sequence into a host cell such as an animal cell or yeast cells incorporating a nucleic acid having the nucleotide sequence introduced directly without using a vector and the like as described in 5 above can also be used.
  • a method comprising using a change in the expression level of a nucleic acid such as an micro-RNA or precursor thereof to be targeted in the screening as an index, as well as a method comprising using a change in the expression level of an mRNA having a target sequence for a nucleic acid such as a micro-RNA or a gene product encoded thereby as an index can be mentioned.
  • a test substance is brought into contact with a cell that expresses the nucleic acid, and with a change in the expression level of the nucleic acid selected as an index, a substance that promotes or suppresses the nucleic acid such as a micro-RNA and precursor thereof is obtained.
  • the expression level of a nucleic acid can be detected by the method described in 3 above.
  • a test substance is brought into contact with a cell that expresses the mRNA, and with a change in the expression level of an mRNA having a target sequence of the nucleic acid selected or a gene product encoded thereby as an index, a substance that promotes or suppresses the expression or a function of a nucleic acid such as a micro-RNA and a precursor thereof can be obtained.
  • a DNA having a target sequence of a nucleic acid of the present invention such as micro-RNA inserted into the 3' UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector, a test substance is brought into contact with the cell, and with a change in the expression level of the reporter gene as an index, a substance that promotes or suppresses the expression or a function of a nucleic acid such as a micro-RNA and a precursor thereof can be obtained.
  • Choice of a target sequence can be achieved by the method described in 7 above; as examples of an mRNA having a target sequence of a nucleic acid such as a micro-RNA consisting of the nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741, the above-described genes shown in Table 4 above can be exemplified.
  • Mast cell degranulation control agents comprising a nucleic acid such as a micro-RNA or a micro-RNA precursor
  • Nucleic acids such as micro-RNAs and micro-RNA precursors and nucleic acids having nucleotide sequences complementary to the nucleotide sequences thereof, used in the present invention can be used as mast cell degranulation control agents, that is, degranulation suppressants or degranulation promoters, because they control the expression of genes having a target sequence.
  • nucleic acids (a) to (h) below As active ingredients of mast cell degranulation suppressants, the nucleic acids (a) to (h) below can be mentioned.
  • Substances that promote a function or the expression of the nucleic acids (a) to (h) above can also be used as mast cell degranulation suppressants.
  • Substances that suppress a function or the expression of a target gene for the nucleic acids (a) to (h) above can also be used as mast cell degranulation suppressants.
  • As substances that suppress the expression of a target gene an siRNA against the mRNA of the target gene, an antisense against the target gene, and the like can be mentioned.
  • substances that suppress a function or the expression of the nucleic acids (i) to (p) below can also be used as mast cell degranulation suppressants.
  • substances that suppress a function or the expression of the nucleic acids siRNAs and antisenses against the nucleic acids, and the like can be mentioned.
  • active ingredients of mast cell degranulation promoters the nucleic acids (i) to (p) below can be mentioned.
  • Substances that promote a function or the expression of the nucleic acids (i) to (p) above can also be used as mast cell degranulation promoters.
  • Substances that suppress a function or the expression of a target gene for the nucleic acids (i) to (p) above can also be used as mast cell degranulation promoters.
  • substances that suppress the expression of a target gene an siRNA against the mRNA of the target gene, an antisense against the target gene and the like can be mentioned.
  • substances that suppress a function or the expression of the nucleic acids (a) to (h) above can also be used as mast cell degranulation promoters.
  • substances that suppress a function or the expression of the nucleic acids an siRNA and antisense against the nucleic acids and the like can be mentioned.
  • a mast cell degranulation control agent of the present invention a vector that expresses one of the nucleic acids (a) to (h) and (i) to (p) above can also be used.
  • Diagnostic agents and therapeutic agents containing a nucleic acid such as a micro-RNA or a micro-RNA precursor
  • Nucleic acids, such as micro-RNAs and micro-RNA precursors, used in the present invention can be used as therapeutic agents for diseases resulting from a mast cell abnormality and the like, because they control the expression of genes having a target sequence, or control the expression of nucleic acids, such as micro-RNAs, used in the present invention.
  • siRNAs against a target gene for the nucleic acids can be used as therapeutic agents for diseases resulting from a mast cell abnormality and the like because they control the expression of the gene.
  • mast cell abnormalities abnormalities of mast cell differentiation and degranulation, inflammatory mediator production, cytokine production, chemokine production and the like can be mentioned; as diseases caused thereby, atopic dermatitis, asthma, chronic obstructive lung disease, allergic disease and the like can be mentioned.
  • a diagnostic agent comprising a nucleic acid of the present invention may comprise reagents necessary for quantitation or detection of mutation of_a nucleic acid used in the present invention, for example, buffering agents, salts, reaction enzymes, labeled proteins that bind to_a nucleic acid of the present invention, and a color developer for detection and the like.
  • a therapeutic agent containing as an active ingredient a nucleic acid used in the present invention can be administered alone, the same is normally desirably administered as a pharmaceutical preparation produced by an optionally chosen method known well in the technical field of pharmaceutical making with one or more pharmacologically acceptable carriers blended therein.
  • the route of administration used is desirably the most effective one in treatment; oral administration, or parenteral administration such as intraoral administration, airway administration, intrarectal administration, subcutaneous administration, intramuscular administration and intravenous administration can be mentioned, and desirably intravenous administration can be mentioned.
  • dosage forms sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injection formulations, ointments, tapes and the like can be mentioned.
  • preparations appropriate for oral administration emulsions, syrups, capsules, tablets, powders, granules and the like can be mentioned.
  • Liquid preparations like emulsions and syrups can be produced using water, saccharides such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil, antiseptics such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor and peppermint and the like as additives.
  • saccharides such as sucrose, sorbitol, and fructose
  • glycols such as polyethylene glycol and propylene glycol
  • oils such as sesame oil, olive oil, and soybean oil
  • antiseptics such as p-hydroxybenzoic acid esters
  • flavors such as strawberry flavor and peppermint and the like as additives.
  • Capsules, tablets, powders, granules and the like can be produced using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropylcellulose, and gelatin, surfactants such as fatty acid esters, plasticizers such as glycerin and the like as additives.
  • excipients such as lactose, glucose, sucrose, and mannitol
  • disintegrants such as starch and sodium alginate
  • lubricants such as magnesium stearate and talc
  • binders such as polyvinyl alcohol, hydroxypropylcellulose, and gelatin
  • surfactants such as fatty acid esters
  • plasticizers such as glycerin and the like as additives.
  • injection formulations As appropriate preparations for parenteral administration, injection formulations, suppositories, sprays and the like can be mentioned.
  • An injection formulation is prepared using a carrier consisting of a salt solution, a glucose solution or a mixture of both and the like.
  • a suppository is prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • a spray is prepared using a carrier that does not stimulate the recipient's oral cavity and airway mucosa, and that disperses the active ingredients as fine particles to facilitate the absorption thereof, and the like.
  • the carrier specifically, lactose, glycerin and the like can be exemplified.
  • preparations such as aerosols and dry powders are possible.
  • components exemplified as additives for oral preparations can also be added.
  • the dose or frequency of administration varies depending on desired therapeutic effect, method of administration, duration of treatment, age, body weight and the like, and is normally 10 ⁇ g/kg to 20 mg/kg per day for an adult.
  • a therapeutic agent containing as an active ingredient a nucleic acid used in the present invention can also be produced by blending a vector that expresses the nucleic acid used in the present invention and a base for nucleic acid-based therapeutic agents ( Nature Genet., 8, 42(1994 )).
  • the base used in the therapeutic agent of the present invention may be any base for ordinary use in injection formulations; distilled water, solutions of salts such as sodium chloride or a mixture of sodium chloride and an inorganic salt, solutions of mannitol, lactose, dextran, glucose and the like, solutions of amino acids such as glycine and arginine, mixed solutions of organic acid solutions or salt solutions and glucose solution and the like can be mentioned.
  • an injection formulation may be prepared as a solution, suspension, or dispersion.
  • injection formulations can also be prepared as preparations for dissolution before use, by procedures such as powdering and lyophilization.
  • a therapeutic agent of the present invention can be used for treatment as is in the case of a liquid, or after being dissolved in a base described above, optionally sterilized, in the case of a solid, just before treatment.
  • the recombinant viral vector prepared by the method described in 5 above can be mentioned, more specifically, a retrovirus vector and a lentivirus vector and the like can be mentioned.
  • a viral vector can be prepared.
  • the viral vector is capable of stably reaching the desired cell, being incorporated into cells by endosome, being decomposed in the cells, and efficiently expressing the nucleic acid.
  • a viral vector based on Sendai virus which is a (-) strand RNA virus, has been developed ( WO97/16538 , WO97/16539 ); using the Sendai virus, a Sendai virus vector that expresses a nucleic acid used in the present invention can be prepared.
  • a nucleic acid used in the present invention can also be introduced by a non-viral nucleic acid introduction method. The same can be introduced by, for example, calcium phosphate coprecipitation ( Virology, 52, 456-467 (1973 ); Science, 209, 1414-1422 (1980 )], microinjection method ( Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980 ); Proc. Natl. Acad.
  • Membrane fusion-mediated introduction mediated by liposome allows a nucleic acid used in the present invention to be incorporated locally in the tissue, and to be expressed, by administering a liposome preparation directly to the target tissue ( Hum. Gene Ther., 3, 399 (1992 )).
  • a technology including direct uptake of nucleic acid is preferable.
  • receptor-mediated nucleic acid introduction for example, a method performed by binding a nucleic acid to a protein ligand via polylysine can be mentioned.
  • a ligand is chosen on the basis of the presence of a corresponding ligand receptor on the cell surface of the desired cell or tissue.
  • the ligand-nucleic acid conjugate can be injected directly into a blood vessel as desired, and can be directed to a target tissue wherein receptor binding and nucleic acid-protein complex internalization occur.
  • an adenovirus may be infected simultaneously to destroy the endosome function.
  • a nucleic acid such as a micro-RNA or a micro-RNA precursor exhibits at least one of the actions of activation suppression, degranulation suppression, inflammatory mediator production suppression, cytokine production suppression, and chemokine production suppression on mast cells
  • a nucleic acid used in the present invention or an siRNA against a target gene for the nucleic acid into a mast cell, thereafter stimulating the mast cell, measuring a released substance such as (i) histamine or p-hexosaminidase, which can be an index of degranulation, (ii) an inflammatory mediator such as LTC4, LTD4, LTE4, or PGD2, (iii) a cytokine such as TNF- ⁇ or GM-CSF, or (iv) a chemokine such as IL-8, I-209, or MIP-1 ⁇ , and comparing the result with that obtained when the nucleic acid used in the present invention, or the siRNA against the target gene for the nucleic
  • a nucleic acid, micro RNA or micro-RNA precursor used in the present invention, or an siRNA for a target gene of the micro-RNA into a mast cell, and detecting the induction of apoptosis by a measurement of the fragmentation of chromatin DNA, the TUNEL method and the like, the fact that the siRNA possesses apoptosis inducing action can be confirmed.
  • the fact can also be confirmed by introducing a nucleic acid used in the present invention or an siRNA against a target gene for the nucleic acid into a mast cell in the presence of a substance that suppresses the activation of mast cells, thereafter stimulating the mast cell, and comparing the result with that obtained when the nucleic acid used in the present invention or the siRNA against a target gene for the nucleic acid is not introduced.
  • a method wherein an anti-IgE antibody is added after the cell is cultured with the addition of IgE a method wherein Compound 48/80 is added, a method wherein polymyxin B is added, a method wherein dextran is added, a method wherein a calcium ionophore is added, a method wherein acetylcholine is added, a method wherein carbachol is added, a method wherein thrombin is added, a method wherein concanavalin A is added, a method wherein a calcium ionophore is added, a method wherein ATP is added, a method wherein doxorubicin is added, and the like can be mentioned.
  • substances that suppress the activation of mast cells substances that inhibit the process wherein a micro-RNA is biosynthesized from a micro-RNA precursor can be mentioned.
  • Mast cell activation can also be examined by measuring, in place of degranulation, production of cytokines such as TNF- ⁇ and GM-CSF, production of chemokines such as IL-8, 1-309, and MIP-1 ⁇ , production of inflammatory mediators such as LTC4, LTD4, LTE4, and PGD2 and the like [ Blood, 100, 3861(2002 )].
  • cytokines such as TNF- ⁇ and GM-CSF
  • chemokines such as IL-8, 1-309, and MIP-1 ⁇
  • production of inflammatory mediators such as LTC4, LTD4, LTE4, and PGD2 and the like
  • LAD2 a human micro-RNA precursor was introduced into the human mast cell line LAD2, and the influence of the micro-RNA precursor on the degranulation was examined.
  • LAD2 a recently established human mast cell line, is known to well retain the nature of human mast cells ( Leuk. Res., 27, 671 (2003 ); Leuk. Res., 27, 677 (2003 )).
  • LAD2 was obtained from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD 20892-1881, USA), and cultured using Stem Pro-34 medium (manufactured by Invitrogen Company) containing 100 ng/mLSCF.
  • LAD2 was seeded to a 96-well plate at 4 ⁇ 10 3 cells per well, a micro-RNA precursor was introduced using a lipofection method, specifically TransIT-TKO (manufactured by Mirus Company), to obtain a final concentration of 25 nM.
  • the micro-RNA precursors used were human micro-RNA library Ver.1 and Ver.2 synthesized at Ambion Company. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 ⁇ g/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO 2 concentration.
  • a rabbit antihuman IgE antibody (manufactured by DAKO Company) was added to obtain a final concentration of 10 ⁇ g/mL, and the cells were incubated for 30 minutes in an incubator at 37°C and a 5% CO 2 concentration to induce degranulation. Degrees of degranulation were evaluated by measuring the ⁇ -hexosaminidase activity of the enzyme in the granules released in the medium.
  • a measurement of the ⁇ -hexosaminidase activity was performed by adding to the medium 40 ⁇ L of 20 mmol/L 4-methylumbelliferyl-N-acetyl- ⁇ -D-glucosaminide (manufactured by Sigma Company) dissolved in the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl 2 , 0.6 mmol/L MgCl 2 , 0.6 mmol/L KH 2 PO 4 , 10 mM HERPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), incubating the cells at 37°C for 3 hours, thereafter determining the absorbance at 450 nm using the plate reader EnVision (manufactured by Perkin-Elmer Company).
  • Triton X-100 was added to the medium to obtain a final concentration of 1%, and the same experiment was performed, whereby the total ⁇ -hexosaminidase activity contained in LAD2 was measured.
  • the ratio of degranulation induced by the anti-IgE antibody was calculated as the ratio (%) to the total ⁇ -hexosaminidase activity released with 1% Triton X-100.
  • the relative activity of degranulation was evaluated by the deviation from the median by a calculation of subtracting the median from the degranulation efficiency, and dividing the difference by the mean deviation. Degranulation promoting activity was expressed with plus symbols, and degranulation suppressing activity with minus symbols. Two independent experiments were performed; the results are shown in Table 5-1 to Table 5-2.
  • a measurement of degranulation activity using a 6-well plate was also performed.
  • LAD2 was seeded to a 6-well plate at 5 ⁇ 10 5 cells per well, a micro-RNA precursor was introduced using a lipofection method, specifically Gene Silencer (manufactured by Genlantis Company), to obtain a final concentration of 25 nM.
  • the micro-RNA, precursors used were Pre-miRTMmiRNA Precursor Molecules synthesized at Ambion Company. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • the medium was removed via centrifugation, and the plate was washed with the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl 2 , 0.6 mmol/L MgCl 2 , 0.6 mmol/L KH 2 PO 4 , 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), after which the cells were suspended by the addition of 1.5 mL of the Tyrode buffer solution, and the suspension was dispensed to a 96-well plate at 100 ⁇ L per well.
  • the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl 2 , 0.6 mmol/L MgCl 2 , 0.6 mmol/L KH 2 PO 4 , 10 mM HEPES, 5.6 mmol/L D
  • a rabbit antihuman IgE antibody manufactured by DAKO Company
  • DAKO Company a rabbit antihuman IgE antibody
  • the supernatant was recovered via centrifugation, and the ⁇ -hexosaminidase activity in the supernatant was measured to determine the degree of degranulation.
  • the ⁇ -hexosaminidase activity was evaluated by adding to 50 ⁇ L of the recovered supernatant 50 ⁇ L of 4 mmol/L p-nitrophenyl-N-acetyl- ⁇ -glucosaminide (manufactured by Sigma Company) dissolved in 40 mmol/L citrate solution (pH 4.5), and incubating the supernatant at 37°C for 1 hour, thereafter measuring the absorbance of a sample supplemented with 100 ⁇ L of 0.2 mol/L glycine (pH 10.7) at 405 nm using the plate reader 1420 ARVOsx (manufactured by Perkin-Elmer Company).
  • Triton X-100 was added at a final concentration of 1% in place of the rabbit antihuman IgE antibody, and the same experiment was performed, whereby the total ⁇ -hexosaminidase activity in LAD2 was measured.
  • the ratio of degranulation was calculated as the ratio (%) of the ⁇ -hexosaminidase activity in the supernatant to the total ⁇ -hexosaminidase activity, and the relative activity of degranulation was calculated for each precursor with the ratio of degranulation in a negative control plot (Gene Silencer only) taken as 1.0.
  • Each of antisense oligonucleotides against hsa-miR-194, hsa-miR-500 and hsa-miR-365 (these are nucleic acids consisting of the nucleotide sequences of SEQ ID NOs: 67, 68 and 147, respectively) was introduced into LAD2 using a lipofection method to obtain a final concentration of 25 nM.
  • the micro-RNA antisense oligonucleotides used were Anti-miRTMmiRNA Inhibitors (manufactured by Ambion Company). Lipofection was performed according to the method described in the instruction manual attached to the product.
  • human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 ⁇ g/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO 2 concentration.
  • a rabbit antihuman IgE antibody (manufactured by DAKO Company) was added to obtain a final concentration of of 10 ⁇ g/mL, and the cells were incubated for 30 minutes in an incubator at 37°C and a 5% CO 2 concentration to induce degranulation.
  • Degrees of degranulation were evaluated by measuring the ⁇ -hexosaminidase activity of the enzyme in the granules released into the medium.
  • the measurement of the ⁇ -hexosaminidase activity was performed by adding to the medium 40 ⁇ L of 20 mmol/L 4-methylumbelliferyl-N-acetyl- ⁇ -D-glucosaminide (manufactured by Sigma Company) dissolved in the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl, 0.6 mmol/L MgCl 2 , 0.6 mmol/L KH 2 PO 4 , 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), and incubating the cells at 37°C for 3 hours, thereafter determining the absorbance at 450 nm using the plate reader Envision (manufactured by Perkin-
  • Triton X-100 was added to the medium to obtain a final concentration of 1%, and the same experiment was performed, whereby the total ⁇ -hexosaminidase activity contained in LAD2 was measured. The mean for the wells with no cells seeded thereto was calculated, and this was subtracted as the background from each measured value.
  • the ratio of degranulation induced by the anti-IgE antibody was calculated as the ratio (%) of the ⁇ -hexosaminidase activity to the total ⁇ -hexosaminidase activity released with 1% Triton X-100.
  • the relative activity of degranulation was evaluated by the deviation from the median by a calculation of subtracting the median from the degranulation efficiency, and dividing the difference by the mean deviation. Degranulation promoting activity was expressed with plus symbols, and degranulation suppressing activity with minus symbols. Two independent experiments were performed; the results are shown in Table 8.
  • human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 ⁇ g/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO 2 concentration; the following day, a rabbit antihuman IgE antibody was added to induce degranulation, and the ratio of degranulation was measured.
  • Table 9 The results are shown in Table 9.
  • Dicerl-siRNA siRNA of the Dicerl gene
  • a human micro-RNA precursor were co-transfected to LAD2, and the influence of the micro-RNA precursor on the degranulation stimulated by Compound 48/80 was examined.
  • LAD2 was seeded to a 96-well plate at 2 ⁇ 10 4 cells per well, and the Dicerl-siRNA and the micro-RNA precursor were co-transfected using a lipofection method, specifically GeneSilencer (manufactured by Genlantis Company), to obtain a final concentration of 25 nM of each.
  • the sequence of the siRNA against the human Dicerl gene used was the Dicerl-siRNA that targets SEQ ID NO:4148 (manufactured by QIAGEN Company).
  • the micro-RNA precursor used was a human micro-RNA library synthesized at Ambion Company.
  • siRNA allstars As negative controls for the siRNA and the micro-RNA precursor, All Stars Negative Control siRNA (hereinafter, siRNA allstars) (manufactured by QIAGEN Company) and Pre-miRTMmiRNA Precursor Negative Control #1 (hereinafter, miR-negacon#1) (manufactured by Ambion Company), respectively, were provided, and were introduced into LAD2 in the same manner. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • the culture medium was replaced with the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl 2 , 0.6 mmol/L MgCl 2 , 0.6 mmol/L KH 2 PO 4 , 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), and the cells were suspended.
  • the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl 2 , 0.6 mmol/L MgCl 2 , 0.6 mmol/L KH 2 PO 4 , 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4
  • Degrees of degranulation were evaluated by measuring the ⁇ -hexosaminidase activity of the enzyme in the granules released into the culture supernatant.
  • the ⁇ -hexosaminidase activity was evaluated by adding to the recovered culture supernatant 50 mL of 4 mmol/L 4-nitrophenyl-N-acetyl- ⁇ -D-glucosaminide (manufactured by Sigma-Aldrich Company) dissolved in 40 mmol/L citric acid buffer solution (pH 4.5), and incubating the supernatant at 37°C for 1 hour, thereafter determining the absorbance of a sample supplemented with 100 mL of 0.2 mol/L glycine (pH 10.7) at 405 nm using the plate reader 1420 ARVOsx (manufactured by Perkin-Elmer Company).
  • the ratio of degranulation induced by Compound 48/80 was calculated as the ratio (%) to the ⁇ -hexosaminidase activity in the plot with the addition of 1% Triton X-100.
  • the activity of the micro-RNA for cancelling the degranulation suppression due to introduction of the Dicerl-siRNA was calculated as a relative activity with the ratio of degranulation in the plot with co-transfection of the Dicerl-siRNA and miR-negacon#1 taken as 0%, and with the ratio of degranulation in the plot with co-transfection of the siRNA allstars and micro-RNA-negacon#1 taken as 100%. The results are shown in Table 10.
  • Each of the miR-142-3p and miR-24 precursors of human micro-RNA was introduced into LAD2, and gene clusters the amounts of whose expression fluctuate due to introduction of each of the micro-RNA, precursors were comprehensively searched for using an RNA array.
  • LAD2 was seeded to a 6-well plate at 5 ⁇ 10 5 cells per well, and each micro-RNA precursor was introduced using a lipofection method, specifically GeneSilencer (manufactured by Genlantis Company), to obtain a final concentration of 30 nM.
  • the micro-RNA precursors used were Pre-miRTM miRNA Precursor Molecules synthesized at Ambion Company.
  • Pre-miRTMRNA Precursor Negative Control#2 manufactured by Ambion Company (hereinafter, miR-negacon#2) was used. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • the purified total RNA was analyzed for mRNA expression using the Whole Human Genome Oligo Microarray manufactured by Agilent Technologies Company.
  • the analytical procedure was in accordance with the protocol of Agilent Technologies Company. Specifically, a cRNA obtained by labeling the total RNA with Cy3 was hybridized to the microarray at 65°C for about 17 hours, and the microarray was washed, after which the microarray was scanned at a resolution of 5 ⁇ m using the Agilent technologies Microarray Scanner to acquire signal values.
  • a mast cell degranulation control agent a diagnostic agent or therapeutic agent for a disease resulting from mast cell degranulation control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent can be provided. These are useful in the diagnosis or treatment of a disease resulting from mast cell degranulation control.

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Abstract

Provided are a mast cell degranulation control agent, a diagnostic agent or therapeutic agent for a disease resulting from mast cell degranulation control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent, all of which involve the use of a nucleic acid and the like. These are useful in the diagnosis or treatment of a disease resulting from mast cell degranulation control.

Description

    Technical Field
  • The present invention relates to a mast cell degranulation control agent, a diagnostic agent or therapeutic agent for a disease resulting from an abnormality of mast cell degranulation, control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent.
  • Background Art
  • Micro-RNA (miRNA), which is one kind of nucleic acids, is a small non-coding single-stranded RNA of about 22 nucleotides that is not translated into a protein, and has known as being present in many types in organisms, including humans (non-patent documents 1 and 2). A micro-RNA is produced from a gene transcribed to a single or clustered micro-RNA precursor. Specifically, first, a primary-miRNA (pri-miRNA), which is a primary transcript, is transcribed from the gene, then, in stepwise processing from the pri-miRNA to a mature type micro-RNA, a precursor-miRNA (pre-miRNA) of about 70 nucleotides having a characteristic hairpin structure is produced from the pri-miRNA. Furthermore, the mature type micro-RNA is produced from the pre-miRNA by Dicer-mediated processing (non-patent document 3).
  • A mature type micro-RNA is thought to be involved in the post-transcriptional control of gene expression by complementarily binding to an mRNA for a target to suppress the translation of the mRNA, or to degrade the mRNA.
    Although the mechanism in which micro-RNAs suppress the expression of target mRNAs has not been clarified in full, its outline is being elucidated through recent years' research. A micro-RNA suppresses the expression of the mRNA for a target by binding to a partially complementary sequence in the 3'-untranslational region (3'-UTR) of the target mRNA to suppress the translation thereof, or to degrade the target mRNA. Although the complementarity may not be complete, it has been shown that the complementarity of the 2nd to 8th nucleotides from the 5'-end of the micro-RNA is particularly important; this region is sometimes called the "seed sequence" of the micro-RNA (non-patent document 4). It has been shown that micro-RNAs having a same seed sequence suppress the expression of a same target mRNA even if their other sequences differ. Therefore, by making a sequence complementary to a sequence present at the 3'-end of an optionally chosen mRNA as the seed sequence, an RNA having micro-RNA-like activity can be designed. Usually, the term micro-RNA, unlike siRNA, often refers exclusively to an RNA that occurs naturally in cells, so a micro-RNA-like sequence designed as described above is sometimes particularly referred to as an artificial micro-RNA.
  • As of August 2007, in the micro-RNA database miRBase (http://microrna.sanger.ac.uk/), 533 species of micro-RNAs were registered for humans, and 5,071 species for all organisms. Of the micro-RNAs expressed in mammals, including humans, only some have their physiological functions elucidated to date, including miR-181, which is involved in hematopoietic lineage differentiation (non-patent document 5), miR-375, which is involved in insulin secretion (non-patent document 6), and the like; many have their bioactivities unclarified. However, studies using nematodes or Drosophila have shown that micro-RNAs play various important roles in the development and differentiation in organisms, and a report of the relation to human diseases has been reported suggesting a profound relation to cancers (non-patent document 7).
  • Mast cells are known to become activated by various stimuli to undergo degranulation and release or produce many inflammatory mediators (Non-patent Documents 8 to 10). These mediators are diverse and include amines, arachidonic acid metabolites, proteases, cytokines, chemokines and the like. For example, it is known that when an antigen is recognized by a mast cell, histamine and tryptase are quickly released upon degranulation, and chemical mediators such as prostaglandin D2 (PGD2), leukotriene (LT), and platelet activation factor (PAF), various chemokines such as macrophage inflammatory protein (MIP)-1α, and various cytokines such as granulocyte macrophage colony stimulation factor (GM-CSF) are newly synthesized and released. Regarding major basic proteins, which are cytotoxic proteins that have been thought to be produced by eosinophils, it has recently been shown that in the case of humans, they are produced in large amounts by mast cells (non-patent document 11).
  • Hence, mast cells are thought to play major roles in the pathogenesis of various allergic diseases; therefore, it is thought that by controlling a function of mast cells, treatment of allergic diseases is possible.
    Although a wide variety of existing therapeutic agents for allergies are known to possess the action of suppressing the release of inflammatory mediators from mast cells, their studies have traditionally been conducted mainly using rodent mast cells, and there have been no adequate investigations of whether the existing therapeutic agents are actually effective on human mast cells. However, recently, a method of preparing human mast cells has been established, enabling analyses of the actions of existing drugs on human mast cells, and enabling comparisons with their actions on rodent mast cells. As a result, it is known that rodent mast cells and human mast cells have different reactivities to drugs (non-patent document 9). For example, sodium cromoglicate, which was used as a suppressant of inflammatory mediator release, remarkably suppressed the IgE-dependent release of inflammatory mediators in rat abdominal mast cells, but the action thereof on human mast cells was not potent (non-patent document 12). Azelastine hydrochloride, at high concentrations, suppressed the release of histamine, PGD2, and LT and production of GM-CSF and MIP-1α, from human mast cells in culture, but none of these activities were potent. Suplatast tosilate, which was used as an anti-cytokine drug, exhibited inflammatory mediator release suppressive action on rat mast cells, but lacked action on human mast cells (non-patent document 12).
  • Genes expressed in mast cells are important in that they are potential targets of therapeutic agents for a wide variety of allergic diseases; for example, drugs that act on GPCR expressed in mast cells are known to remarkably suppress the production of inflammatory mediators from cultured human mast cells. Specifically, the β2 adrenaline receptor stimulant isoproterenol suppresses the release of histamine, LT, PGD2, GM-CSF and MIP-1α from cultured human mast cells by 80% or more at a low concentration of 10 nmol/l (Non-patent Document 13).
    As a result of a comparison of genes whose expression is induced in human and mouse mast cells activated by various stimuli, it was shown that the genes expressed in humans and mice do not always agree with each other (Non-patent Document 14). Additionally, as stated above, there are also interspecific differences in drug susceptibility between humans and mice.
    In view of the association of micro-RNAs in gene expression control, it is thought that micro-RNAs expressed in mast cells can also be candidates for therapeutic agents for a wide variety of allergic diseases. At present, an analysis of micro-RNAs expressed in mouse marrow-derived mast cells (Non-patent Document 15) is the only available relevant report, with no reports on micro-RNAs expressed in human mast cells. Taking into account the interspecific differences between humans and mice, it is difficult to predict information on the expression of micro-RNAs in human mast cells on the basis of information on the expression of micro-RNAs in mouse mast cells. Additionally, there is no knowledge about the involvement of micro-RNAs in the diverse functions of mast cells.
  • [Conventional Art Documents] [Non-patent Documents]
    • non-patent document 1 : Science, 294, 853-858, (2001)
    • non-patent document 2 : Cell, 113, 673-676, (2003)
    • non-patent document 3: Nature Reviews Genetics, 5, 522-531, (2004)
    • non-patent document 4: Current Biology, 15, R458-R460 (2005)
    • non-patent document 5: Science, 303, 83-86, (2004)
    • non-patent document 6: Nature, 432, 226-230, (2004)
    • non-patent document 7: Nature Reviews Cancer, 6, 259-269, (2006)
    • non-patent document 8: Himan Saibo no Rinsho, ed. Motohito Kurosawa, Sentan Igaku-sha Ltd., p 142 (2001)
    • non-patent document 9: Himan Saibo no Rinsho, ed. Motohito Kurosawa, Sentan Igaku-sha Ltd., p 559 (2001)
    • non-patent document 10: Crit. Rev. Immunol., 22, 115-140 (2002)
    • non-patent document 11: Blood, 98, 1127-1134 (2001)
    • non-patent document 12: Clin. Exp. Allergy, 28, 1228-1236 (1998)
    • non-patent document 13: J. Allergy Clin. Immunol., 103, 421-426 (1999)
    • non-patent document 14: Blood, 100, 3861-3868 (2002)
    • non-patent document 15: Genome Biology, 6, R71 (2005)
    [Summary of the Invention] Problems to Be Solved by the Invention
  • It is expected that by identifying nucleic acids such as micro-RNAs and precursors thereof expressed in various human organs, and analyzing the functions thereof to elucidate their relations to diseases, new therapeutic agents and diagnostic agents will be developed.
    In particular, finding nucleic acids, such as micro-RNAs and precursors thereof, that act in mast cells is expected to lead to the elucidation of functions such as mast cell differentiation and degranulation, cytokine-producing mast cell differentiation and degranulation, inflammatory mediator production, cytokine production, and chemokine production, and hence lead to the development of methods of mast cell isolation, cultivation, differentiation control, degranulation control, inflammatory mediator production control, cytokine production control, and chemokine production control, and new therapies for allergic diseases and the like involving the utilization thereof.
  • It is an object of the present invention to provide a mast cell degranulation control agent, a diagnostic agent or therapeutic agent for a disease resulting from mast cell degranulation control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent, all of which involve the use of a nucleic acid and the like.
  • Means for Solving the Problems
  • The present invention relates to the following (1) to (18).
    1. (1) A mast cell degranulation control agent comprising as an active ingredient any one of the nucleic acids (a) to (h) below:
      1. (a) a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
      2. (b) a nucleic acid of 17 to 28 nucleotides comprising a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
      3. (c) a nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
      4. (d) a nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
      5. (e) a nucleic acid comprising the 2nd to 8th nucleotides of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
      6. (f) a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147,
      7. (g) a nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147, and
      8. (h) a nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147.
    2. (2) The mast cell degranulation control agent according to (1), wherein the nucleic acid is a micro-RNA or a micro-RNA precursor.
    3. (3) A mast cell degranulation control agent comprising as an active ingredient a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid of (1).
    4. (4) A mast cell degranulation control agent comprising as an active ingredient a double-stranded nucleic acid consisting of the nucleic acid of (1) and a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of the former nucleic acid.
    5. (5) A mast cell degranulation control agent comprising as an active ingredient a vector that expresses the nucleic acid of any one of (1) to (3) or the double-stranded nucleic acid of (4).
    6. (6) A mast cell degranulation control agent comprising as an active ingredient a substance that controls the expression or a function of the nucleic acid of (1).
    7. (7) The mast cell degranulation control agent according to (6), wherein the substance that controls the expression or a function of the nucleic acid is an siRNA or an antisense oligonucleotide.
    8. (8) A mast cell degranulation control agent comprising as an active ingredient a substance that suppresses the expression of a target gene for the nucleic acid of (1).
    9. (9) The mast cell degranulation control agent according to (8), wherein the substance that suppresses the expression of a target sequence for a nucleic acid is an siRNA or an antisense oligonucleotide.
    10. (10) A diagnostic agent or therapeutic agent for a disease resulting from an abnormality of mast cells, comprising as an active ingredient the nucleic acid of any one of (1) to (3), the double-stranded nucleic acid of (4), the vector of (5), or the substance of any one of (6) to (9).
    11. (11) A diagnostic agent for a disease resulting from an abnormality of mast cells, comprising as an active ingredient a reagent for detecting the amount expressed of the nucleic acid of (1), a mutation of the nucleic acid or a mutation of the genome that encodes the nucleic acid.
    12. (12) The diagnostic agent or therapeutic agent according to (10) or (11), wherein the disease resulting from an abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic diseases.
    13. (13) A method for treating a disease resulting from an abnormality of mast cells, comprising administering an effective amount of the degranulation suppressant of any one of (1) to (9) to a subject in need thereof.
    14. (14) The method according to (13), wherein the disease resulting from an abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic diseases.
    15. (15) A use of the degranulation suppressant of any one of (1) to (9) for producing a therapeutic agent for a disease resulting from an abnormality of mast cells.
    16. (16) The use according to (15), wherein the disease resulting from an abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic diseases.
    17. (17) A method of controlling mast cell degranulation, comprising using the nucleic acid of any one of (1) to (3), the double-stranded nucleic acid of (4), the vector of (5), or the substance of any one of (6) to (9).
    18. (18) A screening method for a mast cell degranulation control agent, wherein the promotion or suppression of the expression or a function of the nucleic acid of (1) serves as an index.
    Effect of the Invention
  • According to the present invention, it is possible to provide a mast cell degranulation control agent, a diagnostic agent or therapeutic agent for a disease resulting from mast cell degranulation control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent, all of which involve the use of a nucleic acid and the like.
  • [Mode for Carrying out the Invention]
  • The nucleic acid used in the present invention may be any molecule, as far as it is a molecule resulting from polymerization of a nucleotide or a molecule functionally equivalent to the nucleotide; for example, an RNA, which is a ribonucleotide polymer, a DNA, which is a deoxyribonucleotide polymer, a mixed polymer of RNA and DNA, and a nucleotide polymer, including a nucleotide analogue, can be mentioned; furthermore, the nucleic acid may be a nucleotide polymer, including a nucleic acid derivative. In addition, the nucleic acid in the present invention may be a single-stranded nucleic acid or a double-stranded nucleic acid. Double-stranded nucleic acids include double-stranded nucleic acids wherein one strand hybridizes with the other strand under stringent conditions.
  • The nucleotide analogue may be any molecule, as far as it is a molecule prepared by modifying a ribonucleotide, a deoxyribonucleotide, an RNA or a DNA in order to improve the nuclease resistance thereof, to stabilize the same, to increase the affinity thereof for a complementary chain nucleic acid, to increase the cell permeability thereof, or to visualize the same, compared with the RNA or DNA; for example, a nucleotide analogue modified at the sugar moiety thereof, a nucleotide analogue modified at phosphoric acid diester bond and the like can be mentioned.
  • The nucleotide analogue modified at the sugar moiety thereof may be any one, as far as an optionally chosen chemical structural substance has been added to, or substituted for, a portion or all of the chemical structure of the sugar of the nucleotide; for example, a nucleotide analogue substituted by 2'-O-methylribose, a nucleotide analogue substituted by 2'-O-propylribose, a nucleotide analogue substituted by 2'-methoxyethoxyribose, a nucleotide analogue substituted by 2'-O-methoxyethylribose, a nucleotide analogue substituted by 2'-O-[2-(guanidium)ethyl]ribose, a nucleotide analogue substituted by 2'-O-fluororibose, a bridged nucleic acid (BNA) having two cyclic structures as a result of introduction of a bridging structure into the sugar moiety, more specifically a locked nucleic acid (LNA) wherein the oxygen atom at the 2' position and the carbon atom at the 4' position have been bridged via methylene, and an ethylene bridged nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)] can be mentioned, and a peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], an oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and a peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like can also be mentioned.
  • The nucleotide analogue modified at phosphoric acid diester bond may be any one, as far as an optionally chosen chemical substance has been added to, or substituted for, a portion or all of the chemical structure of the phosphoric acid diester bond of the nucleotide; for example, a nucleotide analogue substituted by a phosphorothioate bond, a nucleotide analogue substituted by an N3'-P5' phosphoamidate bond, and the like can be mentioned [SAIBO KOGAKU, 16, 1463-1473 (1997)] [RNAi Method and Antisense Method, Kodansha (2005)].
  • The nucleic acid derivative may be any molecule, as long as it is a molecule prepared by adding another chemical substance to the nucleic acid in order to improve the nuclease resistance thereof, to stabilize the same, to increase the affinity thereof for a complementary chain nucleic acid, to increase the cell permeability thereof, or to visualize the same, compared with the nucleic acid; for example, a 5'-polyamine conjugated derivative, a cholesterol conjugated derivative, a steroid conjugated derivative, a bile acid conjugated derivative, a vitamin conjugated derivative, a Cy5 conjugated derivative, a Cy3 conjugated derivative, a 6-FAM conjugated derivative, a biotin conjugated derivative and the like can be mentioned.
  • As examples of nucleic acids used in the present invention, the following nucleic acids (a) to (k) can be mentioned.
    1. (a) A nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741.
    2. (b) A nucleic acid of 17 to 28 nucleotides comprising a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741.
    3. (c) A nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741.
    4. (d) A nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741.
    5. (e) A nucleic acid comprising the 2nd to 8th nucleotides of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741.
    6. (f) A double-stranded nucleic acid consisting of one of the nucleic acids (a) to (e) and a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid, or a nucleic acid comprising the double-stranded nucleic acid.
    7. (g) A double-stranded nucleic acid consisting of one of the nucleic acids (a) to (e) and a nucleic acid that hybridizes under stringent conditions with the nucleic acid, or a nucleic acid comprising the double-stranded nucleic acid.
    8. (h) A single-stranded nucleic acid having a hairpin structure wherein the double-stranded nucleic acid (f) or (g) is joined via a spacer oligonucleotide consisting of 8 to 28 nucleotides, or a nucleic acid comprising the single-stranded nucleic acid.
    9. (i) A nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147.
    10. (j) A nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147.
    11. (k) A nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147.
  • As the above-described nucleic acids, micro-RNAs are preferably used. A micro-RNA refers to a single-stranded RNA 17 to 28 nucleotides long. The peripheral genome sequence, including the sequence, of a micro-RNA has a sequence capable of forming a hairpin structure, and the micro-RNA can be cut out from either one strand of the hairpin. A micro-RNA complementarily binds to an mRNA serving as a target therefor to suppress the translation of the mRNA or promote the decomposition of the mRNA, thereby mediating the post-translational control of gene expression.
  • As examples of micro-RNAs used in the present invention, human micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs: 1 to 150 and 3372 to 3406 can be mentioned. Furthermore, as micro-RNAs having the same function as the function of human micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406, nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs: 151 to 1237 and 3407 to 3684, which are orthologues of the human micro-RNAs, can be mentioned. As specific examples of orthologues of the human micro-RNA of SEQ ID NO:1, those consisting of a nucleotide sequence of any one of SEQ ID NOs:151 to 173 and 3407 can be mentioned. Tables of the correspondence of micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406 and orthologues thereof are shown in Table 1-1 to Table 1-8. The biological species shown in the uppermost fields of Table 1-1 to Table 1-8 are as follows: hsa, Homo sapiens, human being; mmu, Mus musculus, mouse; rno, Rattus norvegicus, rat; xla, Xenopus laevis, African clawed toad; xtr, Xenopus tropicalis, tropical clawed toad; gga, Gallus gallus, chicken; cfa, Canis familiaris, dog; mdo, Monodelphis domestica, gray short-tailed opossum; age, Ateles geoffroyi, black-handed spider monkey; 11a, Lagothrix lagotricha, Humboldt's wooly monkey; sla, Saguinus labiatus, red-bellied tamarin; mml, Macaca mulatta, rhesus monkey; mne, Macaca nemestrina, pig-tailed macaque; ggo, Gorilla gorilla, gorilla; ppa, Pan paniscus, bonobo; ptr, Pan troglodytes, chimpanzee; ppy, Pongo pygmaeus, orangutan; lca, Lemur catta, ring-tailed lemur; cgr, Cricetulus griseus, Chinese hamster; bta, Bos taurus, cattle; oar, Ovis aries, sheep; ssc, Sus scrofa, swine; dre, Danio rerio, zebrafish; fru, Fugu rubripes, globefish; tni, Tetraodon nigroviridis, green spotted puffer. Because humanderived micro-RNAs and orthologues thereof have high sequence identity, they are considered to possess similar functions. Also because micro-RNAs in the same subgroup as classified by alphabetical figures added to the ends of their names also share high sequence identity, they are considered to possess similar functions.
  • [Table 1-1]
    sequence number human miRNA orthologue sequence number
    mmu rno cgr age lla sla mml mne pbi ggo ppa ptr ppy
    seqNo miRNAname mmu rno cgr age lla sla mml mne pbi ggo ppa ptr ppy
    1 hsa-miR-16 151 152 153 154 155 156 157 158 159 160 161
    2 hsa-miR-195 174 175 176 177 178
    3 hsa-miR-17-3p 3408 181 182 183 184 185 186 187 188 189 190
    196 201
    4 hsa-miR-18a 217 197 198 199 200 218 202 203 204 205 206
    5 hsa-miR-18b
    3412 224 228
    6 hsa-miR-20b 223 3413 225 226 227 229 230 231 232 233 234
    7 hsa-miR-21 250 251 252 253 254 255 256 257 258 259
    8 hsa-miR-24 268 269 270 271 272 273 274 275
    9 hsa-miR-25 285 286 287 288 289 290 291 292 293
    10 hsa-miR-32 301 302 303 304 305 306 307 308 309
    3415 315 318
    11 hsa-miR-26b 314 316 317 319 320 321 322 323 324
    3418
    3416 336
    12 hsa-miR-30a-3p 3417 337 338 339 340 341 342
    347
    348 349 354
    13 hsa-miR-34a 3419 350 351 352 353 355 356 357 358 359 360
    372
    380 375
    14 hsa-miR-449 373 374 381
    15 hsa-miR-449b
    16 hsa-miR-107 382 383 384 385 386 387 388 389 390
    17 hsa-miR-107 400
    3420 405
    18 hsa-miR-148b 404 3421 406
    418 419 421
    19 hsa-miR-190 3422 420 422 423 424 425
    3423 433
    20 hsa-miR-199a 3424 3425 431 432 3426 434 435 436 437 438
    21 hsa-miR-199b
    22 hsa-miR-202*
    3429 450
    23 hsa-miR-208 448 449 451
    24 hsa-miR-210 454 455 456
    25 hsa-miR-211 461 462 463 464 465
    26 hsa-miR-214 467 468 469 470 471 472 473 474 475 476
    27 hsa-miR-218 484 485 486 487 488 489 490 491 492 493 494
    28 hsa-miR-299-5p 3431 3432 506
    29 hsa-miR-325 3433 3434 507
    30 hsa-miR-328 509 510
    31 hsa-miR-329 512 513 514
    32 hsa-miR-338 3435 515 3436
    33 hsa-miR-345
    34 hsa-miR-425-5p 3437 3438 3439
    35 hsa-miR-484 523 524 525
    36 hsa-miR-485-5p 3441 3442 527
    37 hsa-miR-486 528 3444
    38 hsa-miR-488 3445
    39 hsa-miR-510
    40 hsa-miR-515-3p
    41 hsa-miR-515-5p
    42 hsa-miR-517*
    3446
    529
    530
    3447
    43 hsa-miR-520d 531
    44 hsa-miR-520f 532
    3448
    45 hsa-miR-520h 533
    46 hsa-miR-522 534
    47 hsa-miR-525*
    48 hsa-miR-573 535
    49 hsa-miR-587
    50 hsa-miR-593
  • [Table 1-2]
    sequence number human miRNA orthologue sequence number
    mmu rno cgr age lla sla mml mne pbi ggo ppa ptr ppy
    51 hsa-miR-595
    52 hsa-miR-604
    53 hsa-miR-612
    54 hsa-miR-625
    55 hsa-miR-634
    56 hsa-miR-635
    57 hsa-miR-637
    58 hsa-miR-647
    3449
    536
    59 hsa-miR-650 537
    60 hsa-miR-654 3450
    61 hsa-miR-658
    62 hsa-miR-660 538
    63 hsa-miR-668 541 542
    64 hsa-miR-675 3451 543
    65 hsa-miR-765 544
    66 hsa-miR-766
    67 hsa-miR-194 545 546 547 548 549 550 551 552
    68 hsa-miR-500
    69 hsa-miR-197 560 561 562 563 564 565 566
    70 hsa-miR-221 568 569 570 571 572 573
    582 596
    583 589 597
    584 590 598
    585 591 599
    586 592 600
    587 593 601
    3453 594 602
    71 hsa-let-7e 588 595 603
    72 hsa-miR-1 669 670 671 672
    73 hsa-miR-206 681 682 683 684 685
    74 hsa-miR-613
    75 hsa-miR-9 691 692 693 694 695 696 697 698
    3458 708 712 716 718 720
    76 hsa-miR-23b 707 709 710 711 713 714 715 717 719 721
    3459 740 744
    77 hsa-miR-27b 739 741 742 743 745 746 747 748 749 750
    78 hsa-miR-28 774 775 776 777 778 779 780 781 782 783 784
    79 hsa-miR-31 786 787 788 789 790 791
    794
    80 hsa-miR-33b 3461 793 795 796 797 798 799 800
    81 hsa-miR-96 805 806 807 808 809 810 811 812
    82 hsa-miR-100 818 879 820 821 822 823 824 825 826 827
    834 836 839 841 843 845 847 849 851
    83 hsa-miR-106a 3462 835 837 838 840 842 844 846 848 850 852
    84 hsa-miR-126* 3463 858
    85 hsa-miR-127 863 864 865 866 867 868 869 870 871
    877
    86 hsa-miR-128b 3465 874 875 876 878 879 880 881
    87 hsa-miR-129 3466 891 3467
    88 hsa-miR-133a 898 899 900 901 902 903 904
    89 hsa-miR-133b
    3469 914 918
    90 hsa-miR-135b 913 915 916 917 919 920 921 922 923
    91 hsa-miR-136 936 937 938 939 940 941 942
    92 hsa-miR-142-5p 946 947 948
    955 956 957
    93 hsa-miR-146a 961 962 3472
    94 hsa-miR-146b
    967 970 977
    3474 971 978 983 986 989
    968 972 974 979 981 984 987 990 992
    95 hsa-miR-181a 969 973 975 976 980 982 985 988 991 993
    96 hsa-miR-182*
    97 hsa-miR-183 1019 1020 1021 1022 1023 1024 1025 1026
    98 hsa-miR-187 1035 1036 1037 1038 1039 1040 1041
    99 hsa-miR-192 1048 1049 1050
    100 hsa-miR-215 1057 1058 1059 1060 1061 1062 1063
  • [Table 1-3]
    number sequence human miRNA orthologue sequence number
    mmu rno cgr age lla sla mml mne pbi ggo ppa ptr ppy
    1067
    101 hsa-miR-200a* 3477
    102 hsa-miR-216 3478 1069 1070 1071 1072 1073
    103 hsa-miR-217 1082 1083 1084 1085
    104 hsa-miR-220 1093 1094 1095
    105 hsa-miR-222 1096 1097 1098 1099
    106 hsa-miR-223 1106 1107 1108 1109 1110 1111 1112 1113
    107 hsa-miR-224 1120 1121 1122 1123 1124 1125 1126 1127
    108 hsa-miR-296 3480 3481 3482
    1130
    109 hsa-miR-302b* 3483
    110 hsa-miR-302c*
    111 hsa-miR-362 3484 3485
    112 hsa-miR-373*
    1135
    113 hsa-miR-374 1133 1134 1136
    1140
    114 hsa-miR-376a 3487 1139 1141
    115 hsa-miR-378 3488 3489
    116 hsa-miR-409-3p
    117 hsa-miR-409-5p 1143 1144 1145
    118 hsa-miR-429 1146 1147 1148
    3490 1157
    119 hsa-miR-200c 1156 1158
    120 hsa-miR-432*
    121 hsa-miR-448 1170 1171 1172
    122 hsa-miR-450 3491
    123 hsa-miR-451 1175 1176 1177
    124 hsa-miR-452 1182
    125 hsa-miR-487b 1183 1184 1185
    126 hsa-miR-489
    127 hsa-miR-514 1192 1193 1194 1195
    128 hsa-miR-517c 3493
    3494
    1197
    129 hsa-miR-518c 1198
    130 hsa-miR-524*
    131 hsa-miR-542-3p 1199 1200 1201
    132 hsa-miR-544 1202 1203
    133 hsa-miR-545
    134 hsa-miR-550
    135 hsa-miR-552
    136 hsa-miR-596
    137 hsa-miR-601 1204
    138 hsa-miR-608
    139 hsa-miR-609 1205
    140 hsa-miR-617
    141 hsa-miR-627
    142 hsa-miR-632 1206
    143 hsa-miR-644 1207
    144 hsa-miR-659
    145 hsa-miR-769-5p
    146 hsa-miR-801
    147 hsa-miR-365 1208 1209 1210
    148 hsa-miR-142-3p 1219 1220 1221
    149 hsa-miR-200a 1224 1225 1226
    150 hsa-miR-381 1234 1235 3498
  • [Table 1-4]
    sequence number human miRNA orthologue sequence number
    mmu rno cgr age lla sla mml mne pbi ggo ppa ptr ppy
    3499 3502
    3500 3503 3508
    3372 hsa-miR-29a 3501 3504 3505 3506 3507 3509 3510 3511 3512 3513 3514
    3373a hsa-miR-29b
    3537 3542 3550
    3538 3543 3551 3564
    3539 3544 3552 3555 3558 3561 3565
    3540 3545 3548 3553 3556 3559 3562 3566
    3374 hsa-miR-30a-5p 3541 3546 3547 3549 3554 3557 3560 3563 3567 3568
    3375 hsa-miR-30b
    3376 hsa-miR-105 3601 3602 3603 3604 3605 3606 3607 3608
    3377 hsa-miR-124a 3611 3612 3613 3614 3615 3616 3617
    877
    3378 hsa-miR-128a 3465 874 875 876 878 879 880 881
    3379 hsa-miR-150 3624 3625 3626
    3380 hsa-miR-154 3631 3632 3633 3634 3635 3636 3637 3638
    3381 hsa-miR-299-3p 3639 3640
    3382 hsa-miR-380-5p
    3383 hsa-miR-383 3642 3643 3644
    3384 hsa-miR-411 3649 3650 3651
    3385 hsa-miR-423 3652 3653 3654
    3386 hsa-miR-433 3655 3656 3657
    3387 hsa-miR-454-3p, 3659
    3388 hsa-miR-501 3662 3663 3664
    3389 hsa-miR-504 3665 3666
    3390 hsa-miR-506 3667 3668 3669
    3391 hsa-miR-507 3671 3672 3673
    3392 hsa-miR-508 3675
    3393 hsa-miR-511
    3394 hsa-miR-512-3p 3677
    3395 hsa-miR-527
    3396 hsa-miR-562 3678
    3397 hsa-miR-567
    3398 hsa-miR-589
    3399 hsa-miR-597 3679
    3400 hsa-miR-605 3680
    3401 hsa-miR-619
    3402 hsa-miR-629
    3403 hsa-miR-640 3681
    3404 hsa-miR-767-5p 3682
    3405 hsa-miR-770-5p 3683
    3406 hsa-miR-802 3684
  • [Table 1-5]
    sequence number human miRNA orthologue sequence number
    ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    166 169
    167 170
    1 hsa-miR-16 162 163 164 165 3407 168 171 172 173
    2 hsa-miR-195 179 180
    3 hsa-miR-17-3p 191 3409 192 193 194 195 3410
    213
    209 210 211 222
    4 hsa-miR-18a 207 208 219 3411 220 221 212 214 215 216
    5 hsa-miR-18b
    238 241 243 246
    6 hsa-miR-20b 235 236 237 239 240 242 244 245 247 248 249
    7 hsa-miR-21 260 261 262 263 264 265 266 267
    3414
    8 hsa-miR-24 276 277 278 279 280 281 282 283 284
    9 hsa-miR-25 294 295 296 297 298 299 300
    10 hsa-miR-32 310 311 312 313
    325 329 332
    11 hsa-miR-26b 326 327 328 330 331 333 334 335
    12 hsa-miR-30a-3p 343 344 345 346
    367
    361 364 368 370
    13 hsa-miR-34a 362 363 365 366 369 371
    14 hsa-miR-449 376 377 378 379
    15 hsa-miR-449b
    16 hsa-miR-107 391 392 393 394 395 396 397 398 399
    17 hsa-miR-140 401 402 403
    407 410 412
    18 hsa-miR-148b 408 409 411 413 414 415 416 417
    427
    19 hsa-miR-190 426 428 429 430
    439 3428
    20 hsa-miR-199a 443 3427 444 445 440 441 442
    21 hsa-miR-199b
    22 hsa-miR-202* 446 447
    23 hsa-miR-208 452 453
    24 hsa-miR-210 457 458 459 460
    25 hsa-miR-211 466
    26 hsa-miR-214 477 478 479 480 481 482 483
    3430 502 504
    27 hsa-miR-218 495 496 497 498 499 500 501 503 505
    28 hsa-miR-299-5p
    29 hsa-miR-325 508
    30 hsa-miR-328 511
    31 hsa-miR-329
    32 hsa-miR-338 516 517 518 519 520 521
    33 hsa-miR-345
    34 hsa-miR-425-5p 3440 522
    35 hsa-miR-484 526
    36 hsa-miR-485-5p 3443
    37 hsa-miR-486
    38 hsa-miR-488
    39 hsa-miR-510
    40 hsa-miR-515-3p
    41 hsa-miR-515-5p
    42 hsa-miR-517*
    43 hsa-miR-520d
    44 hsa-miR-520f
    45 hsa-miR-520h
    46 hsa-miR-522
    47 hsa-miR-525*
    48 hsa-miR-573
    49 hsa-miR-587
    50 hsa-miR-593
  • [Table 1-6]
    sequence number human miRNA orthologue sequence number
    ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    51 hsa-miR-595
    52 hsa-miR-604
    53 hsa-miR-612
    54 hsa-miR-625
    55 hsa-miR-634
    56 hsa-miR-635
    57 hsa-miR-637
    58 hsa-miR-647
    59 hsa-miR-650
    60 hsa-miR-654
    61 hsa-miR-658
    62 hsa-miR-660 539 540
    63 hsa-miR-668
    64 hsa-miR-675
    65 hsa-miR-765
    66 hsa-miR-768
    3452
    67 hsa-miR-194 553 554 555 556 557 558
    68 hsa-miR-500 559
    69 hsa-miR-197 567
    70 hsa-miR-221 574 575 576 577 578 579 580 581
    643
    616 644
    617 632 645 653 661
    618 625 633 646 654 662
    604 610 619 626 634 647 655 663
    605 611 620 627 635 648 856 664
    606 612 621 628 636 649 657 665
    607 613 622 629 637 640 650 658 666
    608 614 623 630 638 641 651 659 667
    71 hsa-let-7e 609 615 624 631 639 642 652 660 668
    675 3454
    72 hsa-miR-1 673 674 676 677 678 679 680
    73 hsa-miR-206 686 687 688 689 690
    74 hsa-miR-613
    702
    3455 3456
    75 hsa-miR-9 699 700 701 703 3457 704 705 706
    723 725 728 730 733 735 737
    76 hsa-miR-23b 722 724 726 727 729 731 732 734 736 738
    763
    764
    757 765 768 771
    752 754 758 760 786 769 772
    77 hsa-miR-27b 751 753 755 756 759 761 762 767 770 773
    78 hsa-miR-28 785
    79 hsa-miR-31 3460 792
    803
    80 hsa-miR-33b 801 802 804
    81 hsa-miR-96 813 814 815 816 817
    82 hsa-miR-100 828 829 830 831 832 833
    83 hsa-miR-106a 853 854 855 856 857
    84 hsa-miR-126≠ 3464 859 860 861 862
    85 hsa-miR-127 872 873
    86 hsa-miR-128b 882 883 884 885 886 887 888 889 890
    87 hsa-miR-129 892 893 894 895 896 897
    909
    906 912
    911 3468
    88 hsa-miR-133a 905 907 908 910
    89 hsa-miR-133b
    929
    924 930 932 934
    90 hsa-miR-135b 925 926 927 928 931 933 935
    91 hsa-miR-136 943 944 945
    3471 951 953
    92 hsa-miR-142-5p 949 3470 950 952 954
    958 959 3473 960
    93 hsa-miR-146a 963 964 965 966
    94 hsa-miR-146b
    994
    995 998 1005 1010
    996 999 1001 1003 1006 1008 1011 1013 1015
    95 hsa-miR-181a 997 1000 1002 1004 1007 1009 1012 1014 1016
    96 hsa-miR-182* 1017 1018 3475 3476
    97 hsa-miR-183 1027 1028 1029 1030 1031 1032 1033 1034
    98 hsa-miR-187 1042 1043 1044 1045 1046 1047
    99 hsa-miR-192 1051 1052 1053 1054 1055 1056
    100 hsa-miR-215 1064 1065 1066
  • [Table 1-7]
    sequence number human miRNA orthologue sequence number
    ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    101 hsa-miR-200a* 1068
    102 hsa-miR-216 1074 1075 1076 1077 1078 1079 1080 1081
    103 hsa-miR-217 1086 1087 1088 1089 1090 1091 1092
    104 hsa-miR-220
    105 hsa-miR-222 3479 1100 1101 1102 1103 1104 1105
    106 hsa-miR-223 1114 1115 1116 1117 1118 1119
    107 hsa-miR-224 1128 1129
    108 hsa-miR-296
    109 hsa-miR-302b* 1131
    110 hsa-miR-302c* 1132
    111 hsa-miR-362
    112 hsa-miR-373*
    3486
    113 hsa-miR-374 1137 1138
    114 hsa-miR-376a 1142
    115 hsa-miR-378
    116 hsa-miR-409-3p
    117 hsa-miR-409-5p
    118 hsa-miR-429 1149 1150 1151 1152 1153 1154 1355
    1160 1164 1166
    119 hsa-miR-200c 1159 1161 1162 1163 1165 1167 1168 1169
    120 hsa-miR-432*
    121 hsa-miR-448 1173
    122 hsa-miR-450 1174
    123 hsa-miR-451 1178 1179 1180 1181
    124 hsa-miR-452
    125 hsa-miR-487b 3492 1186
    126 hsa-miR-489 1187 1188 1189 1190 1191
    127 hsa-miR-514 1196
    128 hsa-miR-517c
    129 hsa-miR-518c
    130 hsa-miR-524*
    131 hsa-miR-542-3p 3495
    132 hsa-miR-544
    133 hsa-miR-545
    134 hsa-miR-550
    135 hsa-miR-552
    136 hsa-miR-596
    137 hsa-miR-601
    138 hsa-miR-608
    139 hsa-miR-609
    140 hsa-miR-617
    141 hsa-miR-627
    142 hsa-miR-632
    143 hsa-miR-644
    144 hsa-miR-659
    145 hsa-miR-769-5p
    146 hsa-miR-801
    147 hsa-miR-365 1211 1212 1213 1214 1215 1216 1217 1218
    148 hsa-miR-142-3p 3496 1222 1223 3497
    149 hsa-miR-200a 1227 1228 1229 1230 1231 1232 1233
    150 hsa-miR-361 1236 1237
  • [Table 1-8]
    sequence number human miRNA orthologue sequence number
    ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    3523
    3515 3520 3524
    3516 3518 3521 3525 3527 3529 3531 3533 3535
    3372 hsa-miR-29a 3517 3519 3522 3526 3528 3530 3532 3534 3536
    3373 hsa-miR-29b
    3574 3579 3584 3590
    3569 3575 3580 3585 3591
    3570 3576 3581 3586 3592 3595 3598
    3571 3577 3582 3587 3593 3596 3599
    3374 hsa-miR-30a-5p 3572 3573 3578 3583 3588 3589 3594 3597 3600
    3375 hsa-miR-30b
    3609
    3376 hsa-miR-105 3610
    3619
    3377 hsa-miR-124a 3618 3620 3621 3622 3623
    3378 hsa-miR-128a 882 883 884 885 886 887 888 889 890
    3379 hsa-miR-150 3627 3628 3629 3630
    3380 hsa-miR-154
    3381 hsa-miR-299-3p
    3382 hsa-miR-380-5p 3641
    3383 hsa-miR-383 3645 3646 3647 3648
    3384 hsa-miR-411
    3385 hsa-miR-423
    3386 hsa-miR-433 3658
    3660
    3387 hsa-miR-454-3p 3661
    3388 hsa-miR-501
    3389 hsa-miR-504
    3390 hse-msR-506 3670
    3391 hsa-miR-507 3574
    3392 hsa-miR-508 3676
    3393 hsa-miR-511
    3394 hsa-miR-512-3p
    3395 hsa-miR-527
    3396 hsa-miR-562
    3367 hsa-miR-567
    3398 hsa-miR-589
    3399 hsa-miR-597
    3400 hsa-miR-605
    3401 hsa-miR-619
    3402 hsa-miR-629
    3403 hsa-miR-640
    3404 hra-miR-767-5p
    3405 hsa-miR-770-5p
    3406 hsa-miR-802
  • Regarding the mechanism in which a micro-RNA suppresses the translation of the mRNA of a target gene therefor, it is known that an mRNA having a nucleotide sequence complementary to the 2nd to 8th nucleotides from the 5'-end of the micro-RNA is recognized as a micro-RNA target gene (Current Biology, 15, R458-R460 (2005)). By this mechanism, the expression of the mRNA is suppressed by the micro-RNA. Therefore, micro-RNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5'-end suppress the expression of the mRNA of the same target gene to exhibit the same function. As micro-RNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5'-end as the nucleotide sequence of a micro-RNA consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406, nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs:1238 to 1543 and 3685 to 3741 can be mentioned. As specific examples of the micro-RNAs, for the micro-RNA of SEQ ID ND:1, micro-RNAs consisting of a nucleotide sequence of any one of SEQ ID NOs:1238 to 1286 can be mentioned. Tables of the correspondence of micro-RNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5'-end as the nucleotide sequence of a micro-RNA consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 150 and 3372 to 3406 are shown in Table 2-1 to Table 2-8. Because micro-RNAs having a common seed sequence are considered to share the same target nucleotide sequence, they are considered to possess similar functions.
  • [Table 2-1]
    sequence number human miRNA sequence of 2nd to 8th on 6'terminal side sequence number of mRNA having sequence with the same 2nd to 8th nucleotides on 5'terminnal side
    hsa mmu rno cgr age lla sla mml mne pbi ggo ppa ptr
    1238 1242 1254
    1239 1243 1246 1255
    1 hsa-miR-16 1240 1244 1247 1249 1251 1256 1258 1260 1262 1264
    2 hsa-miR-195 AGCAGCA 1241 1245 1248 1250 1252 1253 1257 1259 1261 1263 1265
    3 hsa-miR-17-3p CUGCAGU 1287 1288
    4 hsa-miR-18a
    5 hsa-miR-18b AAGGUGC
    6 hsa-miR-20b AAAGUGC
    7 hsa-miR-21 AGCUUAU 1290 1291 1292
    8 hsa-miR-24 GGCUCAG
    1293 1297 1307
    1294 1288 1301 1308
    9 hsa-miR-25 1295 1299 1302 1309
    10 hsa-miR-32 AUUGCAC 1296 1300 1303 1304 1305 1306 1310 1311 1312 1313 1314
    1333
    11 hsa-miR-26b UCAAGUA 1334
    1335
    12 hsa-miR-30a-3p UUUCAGU 1336
    13 hsa-miR-34a
    14 hsa-miR-449
    15 hsa-miR-449b GGCAGUG 1337 1338
    16 hsa-miR-107 OCAGGAU 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348
    17 hsa-miR-140 GUGGUUU
    1359
    18 hsa-miR-148b CAGUGCA 1360 1361 1362 1363
    19 hsa-miR-190 GAUAUGU 1369
    20 hsa-miR-199a
    21 hsa-miR-199b CCAGUGU
    22 hsa-miR-202* UUCCUAU
    23 hsir-miR-208 UAAGACG 1370
    24 hsa-miR-210 UGUGCGU
    25 hsa-miR-211 UCCCUUU 1372 1373 1374 1375 1376 1377 1378 1379 1380
    26 hsa-miR-214 CAGCAGG 1392
    27 hsa-miR-218 UGUGCUU
    28 hsa-miR-299-5p GGUUUAC
    29 hsa-miR-325 CUAGUAG
    30 hsa-miR-328 UGGCCCU
    31 hsa-miR-329 AGACACC 1393 1394 1395
    32 hsa-miR-338 CCAGCAU
    33 hss-miR-395 GCUGACU
    34 hsa-miR-425-5p AUGACAC 1396
    38 hsa-miR-484 CAGGCUC 1398
    36 hsc-miR-4855p GAGGCUG
    37 hsa-miR-486 CCUGUAC
    38 hsa-miR-488 CCAGAUA
    39 hsa-miR-510 ACUCAGG
    1399
    40 hse-miR-515-3p AGUGCCU 1400
    41 hsa-miR-615-5p UCUCCAA 1401
    42 hse-miR-517* CUCUAGA
    1402
    1403
    1404
    1405 1423
    1406 1424
    1407 1413 1425
    1408 1414 1426
    1409 1415 1427
    1410 1416 1428
    1411 1417 1429
    43 hsa-miR-520d AAGUGCU 1412 1418 1419 1420 1421 1422 1430 1431 1432 1433 1434
    44 hsa-miR-520 AGUGCUU 1455
    45 hsa-miR-520h CAAAGUG 1457
    46 hsa-miR-522 AAAUGGU 1458
    47 hsa-miR-525* AAGGCGC 1459
    48 hsa-miR-573 UGAAGUG
    49 hsa-miR-587 UUCCAUA
    50 hsa-miR-593 GGCACCA
  • [Table 2-2]
    sequence number human miRNA sequence of 2nd to miRNA 8th on 5'terminal side sequence number of mRNA having sequence with the same 2nd to 8th nucleotides on 5' terminal side
    hsa mmu mo cgr age lia sla mml mne pbi ggo ppa ptr
    51 hsa-miR-595 AAGUGUG
    52 hsa-miR-604 GGOUGOG
    53 hsa-miR-612 CUGGGCA 1460
    54 hsa-miR-625 GGGGGAA
    55 hsa-miR-634 ACCAGCA
    56 hsa-miR-635 CUUGGGC
    57 hsa-miR-637 CUOGGGG
    58 hsa-miR-647 UGGCUGC
    59 hsa-miR-650 GGAGGCA
    60 hsa-miR-654 GGUGGGC 1461
    61 hsa-miR-658 GCGGAGG
    62 hsa-miR-660 ACCCAUU
    63 hsa-miR-668 GUCACUC
    64 hsa-miR-675 GGUGCGG
    65 hsa-miR-765 GGAGGAG
    66 hsa-miR-766 CUCGAGC
    67 hsa-miR-194 GUAACAG
    68 hsa-miR-500 UGCACCU
    69 hsa-miR-197 UCACCAC
    70 hsa-miR-221 GCUACAU
    1462
    1463
    1464
    1465
    1466
    1467
    1468
    71 hsa-et-7e GAGGUAG 1469 1470 1471 1472 1473 1474 1475 1476
    72 hsa-miR-1
    73 hsa-miR-206
    74 hsa-miR-613 GGAAUGU
    75 hsa-miR-9 CUUUGGU
    1481
    76 hsa-miR-23b UCACAUU 1482
    77 hsa-miR-27b UCACAGU 1483
    78 hsa-miR-28 AGGAGCU 1484 1485 1486
    79 hsa-miR-31 GCAAGAU
    80 hsa-miR-33b UGCAUUG 1488
    81 hsa-miR-96 UUGGGAC 1489 1490
    1492 1494 1496 1499
    82 hsa-miR-100 ACCGGUA 1493 1495 1497 1498 1500 1501 1502 1503 1504
    83 hsa-miR-106a AAAGUGC
    84 hsa-miR-126* AUUAUUA
    85 hsa-miR-127 COGAUCC
    86 hsa-miR-128b CACAGUG 1514
    87 hsa-miR-129 UUUUUGC
    88 hsa-miR-133a
    89 hsa-miR-133b UGGUCCC
    90 hsa-miR-135b AUGGCUU 1516
    91 hsa-miR-136 CUCCAUU
    92 hsa-miR-142-5p AUAAACU
    93 hsa-miR-146a
    94 hsa-miR-146b GAGAACU
    1517
    1518
    95 hsa-miR-181a ACAUUGA 1519
    96 hsa-miR-182* GGUUCUA
    97 hsa-miR-183 AUGGCAC
    98 hsa-miR-187 GGUGUGU
    99 hsa-miR-192
    100 hsa-miR-215 UGACGUA
  • [Table 2-3]
    sequence number human miRNA sequence of 2nd to 8th on 5'terminal side sequence number of mRNA having sequence with the some 2nd to 8th nucleotides on 5'terminal side
    hsa mmu rno cgr age lla sla mml mne pbl ggo ppa ptr
    101 hsa-miR-200a* AUCUUAC 1520
    102 hsa-miR-216 AAUCUCA
    103 hsn-miR-217 ACUGCAU
    104 hsa-miR-220 CACACCG
    105 hsa-miR-222 GCUACAU
    106 hsa-miR-223 GUCAGUU
    107 hsa-miR-224 AAGUCAC
    108 hsa-miR-296 GGGCCCC
    109 hsa-miR-302b* CUUUAAC 1521
    110 hsa-miR-302c* UUAAGAU
    111 hsa-miR-362 AUCCUUG
    112 hsa-miR-373* GUGAAAA 1522 1523
    113 hsa-miR-374 UAUAAUA 1524
    114 hsa-miR-376a UCAUAGA 1525
    115 hsa-miR-378 UCCUGAC
    116 hsa-miR-409 3p GAAUGUU
    117 hsa-miR-409-5p GGUUACC
    118 hsa-miR-429
    119 hsa-miR-200c AAUAGUG 1526 1527
    120 hsa-miR-432* UGGAUGG
    121 hsa-miR-448 UGCAUAU
    122 hsa-miR-450 UUUUGCG
    123 hsa-miR-451 AAGGGUU
    124 hsa-miR-152 GUUUGCA
    125 hsa-miR-487b AUCGUAC
    126 hsa-miR-489 GUGACAU
    127 hsa-miR-514 UUGAGAC
    128 hsa-miR-517c UCGUGCA 1528
    1529
    1530
    1531
    129 hsa-miR-518c AAAGCGC 1532
    1534
    130 hsa-miR-524* UACAAAG 1533 1535
    131 hsa-miR-542-3p GUGAGAG
    132 hsa-miR-544 UUCUGCA
    133 hsa-miR-545 UGAGGAA
    134 hsa-miR-550 GUGUUAC 1536 1537
    135 hsa-miR-552 AGAGGUG
    136 hsa-miR-596 AGGOUGC
    137 hsa-miR-601 GGUCUAG
    138 hsa-miR-608 GGGGUGG
    139 hsa-miR-609 GGGUGUU
    140 hsa-miR-617 GACUUCC
    141 hsa-miR-627 UGAGUCU 1538
    142 hsa-miR-632 UGUGUGC
    143 hsa-miR-644 GUGUGGC
    144 hsa-miR-659 UUGGUUC
    145 hsa-miR-769-5p GAGACCU
    146 hsa-miR-801 AUUGCUC
    147 hsa-miR-365 AAUGCCC
    148 hsa-miR-142-3p GUAGUGU
    149 hsa-miR-200a AACACUG 1539 1540 1541
    150 hsa-miR-361 UAUCAGA
  • [Table 2-4]
    sequence number human miRNA sequence of 2nd to 8th on 5'terminal side sequence number of mRNA having sequence with the same 2nd to 8th nucleotides on 5'terminal side
    hsa mmu rno cgr age lla sla mml mne pbi ggo ppa ptr
    3372 hsa-miR-29a
    3373 hsa-miR-29b AGCACCA 3685
    3686
    3374 hsa-miR-30a-5p 3687
    3375 hsa-miR-30b GUAAACA 3688 3689 3690
    3376 hsa-miR-105 CAAAUGC
    3377 hsa-miR-124a UAAGGCA
    3378 hsa-miR-128a CACAGUG
    3379 hsa-miR-150 CUCCCAA
    3380 hsa-miR-154 AGGUUAU
    3381 hsa-miR-299-3p AUGUGGG
    3382 hsa-miR-380-5p GGUUGAC 3691
    3383 hsa-miR-383 GAUCAGA
    3384 hsa-miR-411 AGUAGAC
    3385 hsa-miR-423 GCUCGGU
    3386 hsa-miR-433 UCAUGAU
    3696
    3692 3697 3701 3705
    3693 3698 3702 3706
    3894 3699 3703 3707
    3387 hsa-miR-454-3p AGUGCAA 3695 3700 3704 3708 3709 3710 3711
    3388 hsa-miR-501 AUCCUUU
    3389 hsa-miR-504 GACCCUG
    3390 hsa-miR-506 AAGGCAC 3732 3733
    3391 hsa-miR-507 UUUGCAC 3738
    3392 hsa-miR-508 GAUUGUA
    3393 hsa-miR-511 UGUCUUU
    3394 hsa-miR-512-3p AGUGCUG 3739
    3395 hsa-miR-527 UGCAAAG 3741
    3396 hsa-miR-562 AAGUAGC
    3397 hsa-miR-567 GUAUGUU
    3398 hsa-miR-589 CAGAACA
    3399 hsa-miR-597 GUGUCAC
    3400 hsa-miR-605 AAAUCCC
    3401 hsa-miR-619 ACCUGGA
    3402 hsa-miR-629 UUCUCCC
    3403 hsa-miR-640 UGAUCCA
    3404 hsa-miR-767-5p GCACCAU
    3405 hsa-miR-770-5p CCAGUAC
    3406 hsa-miR-802 AGUAACA
  • [Table 2-5]
    sequence number human miRNA sequence of 2nd to 8th on 5'terminal side sequence number mTNA having sequence with the same 2nd to 8th nucleotides on 5'terminal side
    ppy ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    1281
    1269 1275 1278 7282
    1 hsa-miR-16 1266 1270 1273 1276 1279 1283
    2 hsa-miR-195 AGCAGCA 1267 7268 1271 1272 1274 1277 1280 1284 1285 1286
    3 hsa-miR-17-3p CUGCAGU 1289
    4 hsa-miR-18a
    5 hsa-miR-18b AAGGUGC
    6 hsa-miR-20b AAAGUGC
    7 hss-miR-21 AGCUUAU
    8 hsa-miR-24 GGCUCAG
    1323
    1317 1324 1328
    9 hsa-miR-25 1318 1321 1325 1329
    10 hsa-miR-32 AUUGCAC 1315 1316 1319 1320 1322 1326 1327 1330 1331 1332
    11 hsa-miR-26b UGAAGUA
    12 hsa-miR-30a-3p UUUCAGU
    13 hsa-miR-34a
    14 hsa-miR-449
    15 hsa-miR-449b GGCAGUG
    16 hsa-miR-107 GCAGCAU 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358
    17 hsa-miR-140 GUGCUUU
    18 hsa-miR-148b CAGUGCA 1364 1365 1366 1367 1368
    19 hsa-miR-190 GAUAUGU
    20 hsa-miR-199a
    21 hsa-miR-199b CCAGUGU
    22 hsa-miR-202* UUCCUAU
    23 hsa-miR-208 UAAGACG 1371
    24 hsa-miR-210 UGUGGGU
    1390
    25 hsa-miR-211 UCCCUUU 1381 1382 1383 1384 1385 1386 1387 1388 1389 1391
    26 hss-miR-214 CAGCAGG
    27 hsa-miR-218 UGUGCUU
    28 hsa-miR-299-5p GGUUUAC
    29 hsa-miR-325 CUAGUAG
    30 hsa-miR-328 UGGCCCU
    31 hsa-miR-329 AGAGACC
    32 hsa-miR- 338 CCAGGAU
    33 hsa-miR-345 GCUGACU
    34 hsa-miR-426-5p AUGACAC 1397
    35 hsa-miR-484 CAGGCUC
    36 hsa-miR-485-5p GAGGCUG
    37 hsa-miR-486 CCUGUAC
    38 hsa-miR-488 CCAGAUA
    39 hsa-miR-510 ACUCAGG
    40 hsa-miR-515-3p AGUGCCO
    41 hsa-miR-516-5p UCUCCAA
    42 hsa-miR-517* GUCUAGA
    1437 1450
    1438 1451
    1439 1442 1447 1452
    1440 1443 1445 1448 1453
    43 hsa-miR-520d AAGUGCU 1435 1436 1441 1444 1446 1449 1454
    44 hsa-miR-520f AGUGCUU 1456
    45 hsa-miR-520h CAAAGUG
    46 hsa-miR-522 AAAUGGU
    47 hsa-miR-525* AAGGCGC
    48 hsa-miR-573 UGAAGUG
    49 hsa-miR-587 UUCCAUA
    50 hsa-miR-593 GGCACCA
  • [Table 2-6]
    sequence number human miRNA sequence of 2nd to 8th on 5'terminal side sequence number of mRNA having sequence with the same 2nd to 8th nucleotides on 5'terminal side
    ppy ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    51 hs a-miR-595 AAGUGUG
    52 hs a-miR-604 GGCUGCG
    53 hs a-miR-612 GUGGGGA
    54 hs a-miR 625 GGGGGAA
    55 hs a-miR-634 ACCAOCA
    56 hs a-miR-635 CUUGGGC
    57 hs a-miR-637 CUGGGGG
    58 hs a-miR-647 UGGCUGC
    59 hs a-miR-650 GGAGGCA
    60 hs a-miR-634 GGUGGGC
    61 hs a-miR-658 GCGGAGG
    62 hs a-miR-660 ACCCAUU
    63 hs a-miR-668 GUCACUC
    64 hs a-miR-675 GGUGCGG
    65 hs a-miR-765 GGAGGAG
    66 hs a-miR-766 CUCCAGC
    67 hs a-miR-194 GUAACAG
    68 hs a-miR-500 UGCACCU
    69 hs a-miR-197 UCACCAC
    70 hs a-miR-221 GCUACAU
    71 hs a-let-7e GAGGUAG 1477 1478 1419 1480
    72 hs a-miR-1
    73 hs a-miR-206
    74 hs a-miR-613 GGAAUGU
    75 hsa-miR-9 CUUUGGU
    76 hsa-miR-23b UCACAUU
    77 hsa-miR-27b UCACAGU
    78 hsa-miR-28 AGGAGCU 1487
    79 hsa-miR-31 GGAAGAU
    80 hsa-miR-33b UGCAUUG
    81 hsa-miR-96 UUGGCAC 1491
    1506 1510
    82 hs a-miR-100 AGCCGUA 1505 3507 150H 1509 1511 1512 1513
    83 hs a-miR-106a AAAGUGC
    84 hs a-miR-126* AUUAUUA
    85 hs a-miR-121 CGGAUCC
    86 hs a-miR-128b GACAGUG
    87 hs a-miR-129 UUUUUGC 1515
    88 hs a-miR-133a
    89 hs a-miR-133 UGGUGCC
    90 hs a-miR-135b AUGGCUU
    91 hs a-miR-136 CUCCAUU
    92 hs a-miR-142-5p AUAAAGU
    93 hs a-miR-146a
    94 hs a-miR-146b GAGAACU
    95 hs a-miR-181a ACAUUCA
    96 hsa-miR-182* GGUUCUA
    97 hsa-miR-183 AUGGCAC
    98 hsa-miR-187 OGUOUCU
    99 hsa-miR-192
    100 hsa-miR-215 UGACCUA
  • [Table 2-7]
    sequence number human miRNA sequence of 2nd to 8th on 5'terminal side sequence number of mRNA having sequence with the same 2nd to 8th nucleotides on 5' terminal side
    ppy ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    101 hs a-miR-200a* AUCUUAC
    102 hs a-miR-216 AAUCUCA
    103 hs a-miR-217 ACUGCAU
    104 hs a-miR-220 CACACCG
    105 hs a-miR-222 GCUACAU
    106 hs a-miR-223 GUCAGUU
    107 hs a-miR-224 AAGUCAC
    108 hs a-miR-296 GGGCCCC
    109 hs a-miR-302b* CUUUAAC
    110 hs a-miR-302c* UUAACAU
    111 hs a-miR-362 AUCCUUG
    112 hs a-miR-373* GUCAAAA
    113 hs a-miR-374 UAUAAUA
    114 hsa-miR-376a UCAUAGA
    115 hsa-miR-378 UCCUGAG
    116 hsa-miR-409-3p GAAUGUU
    117 hsa-miR-409-5p GGUUACC
    118 hsa-miR-429
    119 hsa-miR-200c AAUACUG
    120 hsa-miR-432* UGGAUGG
    121 hsa-miR-448 UGGAUAU
    122 hsa-miR-450 UUUUGCG
    123 hsa-miR-451 AACCGUU
    124 hsa-miR-452 GUUUGCA
    125 hsa-miR-487b AUCGUAC
    126 hsa-miR-489 GUGACAU
    127 hs a-miR-514 UUGACAC
    128 hs a-miR-517c UCGUGCA
    129 hs a-miR-518c AAAGCGC
    130 hs a-miR-524* UACAAAG
    131 hs a-miR-542-3p GUGACAG
    132 hs a-miR-544 UUCUGCA
    133 hs a-miR-545 UCAGCAA
    134 hs a-miR-550 GUCUUAC
    135 hs a-miR-552 ACAGGUG
    136 hs a-miR-596 AGCCUGC
    137 hs a-miR-601 GGUCUAG
    138 hs a-miR-608 GGGGUGG
    139 hs a-miR-609 GGGUGUU
    140 hs a-miR-617 GACUUCC
    141 hs a-miR-627 UGAGUCU
    142 hs a-miR-632 UGUCUGC
    143 hs a-miR-644 GUGUGGC
    144 hs a-miR-659 UUGGUUC
    145 hs a-miR-769-5p GAGACCU
    146 hs a-miR-801 AUUGCUG
    147 hs a-miR-365 AAUGGGC
    148 hs a-miR-142-3p GUAGUGU
    149 hs a-miR-200a AACACUG 1542 1543
    150 hs a-miR-361 UAUCAGA
  • [Table 2-8]
    sequence number human miRNA sequence of 2nd to 8th on 5' terminal side sequence number of mRNA having sequence with the same 2nd to 8th nucleotides on 5' terminal side
    ppy ssy lca cfa mdo gga xla xtr bta oar ssc dre fru tni
    3372 hsa-miR-29a
    3373 hsa-miR-29b AGGACCA
    3374 hsa-miR-30a-5p
    3375 hsa-miR-30b GUAAACA
    3376 hsa-miR-105 CAAAUGC
    3377 hsa-miR-124a UAAGGCA
    3378 hsa-miR-128a CACAGUG
    3379 hsa-miR-150 CUCCCAA
    3380 hsa-miR-154 AGGUUAU
    3331 hsa-miR-299-3p AUGUGGG
    3382 hsa-miR-360-5p GGUUGAC
    3333 hsa-miR-383 GAUGAGA
    3384 hsa-miR-411 AGUAGAC
    3385 hsa-miR-423 GCUCGGU
    3386 hsa-miR-933 UCAUGAU
    3722
    3723
    3713 3729
    3715 3719 3725
    3712 3716 3720 3726 3728 3730
    3387 has-miR-454-3p AGUGCAA 3713 3714 3717 3721 3727 3729 3731
    3388 hsa-miR-501 AUCCUUU
    3389 hsa-miR-504 GACGGUG
    3390 hsa-miR-506 AAGGCAC 3734 3735 3736 3737
    3391 hse-miR-507 UUUGCAC
    3392 hsa-miR-508 GAUUGUA
    3393 hsa-miR-511 UGUCUUU
    3740
    3395 hsa-miR-527 UGCAAAG
    3396 hsa-miR-562 AAGUAGC
    3397 hsa-miR-567 GUAUGUU
    3398 hsa-miR-589 CAGAACA
    3399 hsa-miR-597 GUGUCAC
    3400 hsa-miR-605 AAAUCCC
    3401 hsa-miR-619 ACCUGGA
    3402 hsa-miR-629 UUCUGCG
    3403 hsa-miR-640 UGAUCCA
    3404 hsa-miR-767-5p GGACCAU
    3405 hsa-miR-770-5p GCAGUAC
    3406 hsa-miR-802 AGUAACA
  • As the above-described nucleic acids, micro-RNA precursors are also used preferably. A micro-RNA precursor is a nucleic acid about 50 to about 200 nucleotides long, more preferably about 70 to about 100 nucleotides long, including the above-described nucleic acids used in the present invention, and capable of forming a hairpin structure. A micro-RNA is produced from a micro-RNA precursor via processing by a protein called Dicer.
  • As examples of micro-RNA precursors used in the present invention for the human micro-RNAs of SEQ ID NO:1, nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 1545 can be mentioned. For the micro-RNAs of SEQ ID NOs:2 to 1543 and 3372 to 3741, nucleic acids consisting of a nucleotide sequence of any one of SEQ ID NOs:1546 to 3371 and 3742 to 4147 can be mentioned. Tables of the correspondence of micro-RNAs and micro-RNA precursors used in the present invention are shown in Table 3-1 to Table 3-39.
  • [Table 3-1]
    Sequence number miRNA name sequence number miRNA precisions name
    1 hsa-miR-16 1544 hsa-mir-16-1
    1545 hsa-mir-16-2
    2 hsa-miR-195 1546 hsa-mir-195
    3 hsa-miR-17-3p 1547 hsa-mir-17
    4 hsa-miR-18a 1548 hsa-mir-18a
    5 hsa-miR-18b 1549 hsa-mir-18b
    6 hsa-miR-20b 1550 hsa-mir-20b
    7 hsa-miR-21 1551 hsa-mir-21
    8 hsa-miR-24 1552 hsa-mir-24-1
    1553 hsa-mir-24-2
    9 sa-miR-25 1554 hsa-mir-25
    10 hsa-miR-32 1555 hsa-mir-32
    11 hsa-miR-26b 1556 hsa-mir-26b
    12 hsa-miR-34a-3p 1557 hsa-mir-30a
    13 hsa-miR-34a 1558 hsa-mir-34a
    14 hsa-miR-449 1559 hsa-mir-449
    15 5 hsa-miR-449b 1560 hsa-mir-449b
    16 hsa-miR-107 1561 hsa-mir-107
    17 hsa-miR-140 1562 hsa-mit-140
    18 hsa-miR-148b 1563 hsa-mir-148b
    19 hsa-miR-190 1564 hsa-mir-190
    20 hsa-miR-199a 1565 hsa-mir-199a-1
    1566 hsa-miR-199a-2
    21 hsa-miR-199b 1567 hsa-mir-199b
    22 hsa-miR-202* 1568 hsa-mir-202
    23 hsa-miR-208 1559 hsa-mir-208
    24 hsa-miR-210 1570 hsa-mir-210
    25 hsa-miR-211 1571 hsa-mir-211
    26 hsa-miR-214 1572 hsa-mir-214
    27 hsa-miR-218 1573 hsa-mir-218-1
    1574 hsa-mir-218-2
    28 hsa-miR-299-5p 1575 hsa-mir-299
    29 hsa-miR-325 1576 hsa-mir-325
    30 hsa-miR-328 1577 hsa-mir-328
    31 hsa-miR-328 1578 hsa-mir-329-1
    1579 hsa-mir-329-2
    32 hsa-miR-338 1580 hsa-mir-338
    33 hsa-miR-345 1581 hsa-mir-345
    34 hsa-miR-425-5p 1582 hsa-mir-425
    35 hsa-miR-484 1583 hsa-mir-484
    36 hsa-miR-485-5p 1584 hsa-mir-485
    37 hsa-miR-486 1585 hsa-mir-486
    38 hsa-miR-488 1586 hsa-mir-488
    39 hsa-miR-510 1587 hsa-mir-510
    40 hsa-miR-515-3p 1588 hsa-mir-515-1
    1589 hsa-mir-515-2
    41 hsa-miR-515-5p 1588 hsa-mir-515-1
    1589 hsa-mir-515-2
    42 hsa-miR-517* 1590 hsa-mir-517a
    1591 hsa-mir-517b
    1592 hsa-mir-517c
    43 hsa-miR-520d 1583 hsa-mir-520d
    44 hsa-miR-520f 1594 hsa-mir-520f
    45 hsa-miR-520h 1595 hsa-mir-520h
    46 hsa-miR-522 1596 hsa-mir-522
    47 hsa-miR-525* 1597 hsa-mir-525
    48 hsa-miR-573 1598 hsa-mir-573
    49 hsa-miR-587 1599 hsa-mir-587
    50 hsa-miR-593 1600 hsa-mir-593
  • [Table 3-2]
    sequence number miRNA name sequence number miRNA precursor name
    51 hsa-miR-595 1601 hsa-mir-595
    52 hsa-miR-604 1602 hsa-mir-604
    53 hsa-miR-612 1603 hsa-mir-612
    54 hsa-miR-625 1604 hsa-mir-625
    55 hsa-miR-634 1605 hsa-mir-634
    56 hsa-miR-635 1606 hsa-mir-635
    57 hsa-miR-637 1607 hsa-mir-637
    58 hsa-miR-647 1608 hsa-mir-647
    59 hsa-miR-650 1609 hsa-mir-650
    60 hsa-miR-654 1610 hsa-mir-654
    61 hsa-miR-658 1611 hsa-mir-658
    62 hsa-miR-660 1612 hsa-mir-660
    63 hsa-miR-668 1613 hsa-mir-668
    64 hsa-miR-675 1614 hsa-mir-675
    65 hsa-miR-765 1615 hsa-mir-765
    66 hsa-miR-766 1616 hsa-mir-766
    67 hsa-miR-194 1617 hsa-mir-194-1
    1618 hsa-mir-194-2
    68 hsa-miR-500 1619 hsa-mir-500
    69 hsa-miR-197 1620 hsa-mir-197
    70 hsa-miR-221 1621 hsa-mir-221
    71 hsa-let-7e 1622 hsa-let-7e
    72 hsa-mR-1 1623 hsa-mir-1-1
    1624 hsa-mir-1-2
    73 hsa-miR-206 1625 hsa-mir-206
    74 hsa-miR-613 1626 hsa-mir-613
    75 hsa-miR-9 1627 hsa-mir-9-1
    1628 hsa-mir-9-2
    1629 hsa-mir-9-3
    76 hsa-miR-23b 1630 hsa-mir-23b
    77 hsa-miR-27b 1631 hsa-mir-27b
    78 hsa-miR-28 1632 hsa-mir-28
    79 hsa-miR-31 1633 hsa-mir-31
    80 hsa-miR-33b 1634 hsa-mir-33b
    81 hsa-miR-96 1635 hsa-mir-96
    82 hsa-miR-100 1636 hsa-mir-100
    83 hsa-miR-106a 1637 hsa-mir-106a
    84 hsa-miR-126* 1638 hsa-mir-126
    85 hsa-miR-127 1639 hsa-mir-127
    86 hsa-miR-128b 1640 hsa-mir-128b
    87 hsa-miR-129 1641 hsa-mir-129-1
    1642 hsa-mir-129-2
    88 hsa-miR-133a 1643 hsa-mir-133a-1
    1644 hsa-mir-133a-2
    89 hsa-miR-133b 1645 hsa-mir-133b
    90 hsa-miR-135b 1646 hsa-mir-135b
    91 hsa-miR-136 1647 hsa-mir-136
    92 hsa-miR-142-5p 1648 hsa-mir-142
    93 hsa-miR-146a 1649 hsa-mir-146a
    94 hsa-miR-146b 1650 hsa-mir-146b
    95 hsa-miR-181a 1651 hsa-mir-181a
    96 hsa-miR-182* 1652 hsa-mir-182
    97 hs8-miR-183 1653 hsa-mir-183
    98 hsa-miR-187 1654 hsa-mir-187
    99 hsa-miR-192 1655 hsa-mir-192
    100 hsa-miR-215 1656 hsa-mir-215
  • [Table 3-3]
    sequence number miRNA name sequence number miRNA precursor name
    101 hsa-miR-200a* 1657 hsa-mir-200a
    102 hsa-miR-216 1658 hsa-mir-216
    103 hsa-miR-217 1659 hsa-mir-217
    104 hsa-miR-220 1660 hsa-mir-200
    105 hsa-miR-222 1661 hsa-mir-222
    106 hsa-miR-223 1662 hsa-mir-223
    107 hsa-miR-224 1663 hsa-mir-224
    108 hsa-miR-296 1664 hsa-mir-296
    109 hsa-miR-302b* 1685 hsa-mir-302b
    110 hsa-miR-302c* 1666 hsa-mir-302c
    111 hsa-miR-362 1667 hsa-mir-362
    112 hsa-miR-373* 1668 hsa-mir-373
    113 hsa-miR-374 1669 hsa-mir-374
    114 hsa-miR-376a 1670 hsa-mir-376a-1
    1671 hsa-mir-376a-2
    115 hsa-miR-378 1672 hsa-mir-378
    116 hsa-miR-409-3p 1673 hsa-mir-409
    117 hsa-miR-409-5p 1673 hsa-mir-409
    118 hsa-miR-429 1674 hsa-mir-429
    119 hsa-miR-200c 1675 hsa-mir-200c
    120 hsa-miR-432* 1676 hsa-mir-432
    121 hsa-miR-448 1677 hsa-mir-448
    122 hsa-miR-450 1678 hsa-mir-450-1
    1679 hsa-mir-450-2
    123 hsa-miR-451 1680 hsa-mir-451
    124 hsa-miR-452 1681 hsa-mir-452
    125 hsa-miR-487b 1682 hsa-mir-487b
    126 hsa-miR-489 1683 hsa-mir-489
    127 hsa-miR-514 1684 hsa-mir-514-1
    1685 hsa-mir-514-2
    1888 hsa-mir-514-3
    128 hsa-miR-517c 1592 hsa-mir-517c
    129 hsa-miR-518c 1687 hsa-mir-518c
    130 hsa-miR-524* 1688 hsa-mir524
    131 hsa-miR-542-3p 1689 hsa-mir-542
    132 hsa-miR-544 1690 hsa-mir-544
    133 hsa-miR-545 1691 hsa-mir-545
    134 hsa-miR-550 1692 hsa-mir-550-1
    1693 hsa-mir-550-2
    135 hsa-miR-552 1694 hsa-mir-552
    136 hsa-miR-596 1695 hsa-mir-596
    137 hsa-miR-601 1696 hsa-mir-601
    138 hsa-miR-608 1697 hsa-mir-608
    139 hsa-miR-609 1698 hsa-mir-609
    140 hsa-miR-817 1699 hsa-mir-617
    141 hsa-miR-627 1700 hsa-mir-627
    142 hsa-miR-632 1701 hsa-mir-632
    143 hsa-miR-644 1702 hsa-mir-644
    144 hsa-miR-659 1703 hsa-mir-659
    145 hsa-miR- 769-5p 1704 hsa-mir-789
    146 hsa-miR-801 1705 hsa-mir-801
    147 hsa-miR-365 1706 hsa-mir-365-1
    1707 hsa-mir-365-2
    148 hsa-miR-142-3p 1648 hsa-mir-142
    149 hsa-miR-200a 1657 hsa-mir-200a
    150 hsa-miR-361 1708 hsa-mir-361
  • [Table 3-4]
    sequence number miRNA name sequence number miRNA precursor name
    151 mmu-miR-16 1709 mmu-mir-16-1
    1710 mmu-mir-16-2
    152 mo-miR-16 1711 mo-mir-16
    153 age-miR-16 1712 age-mir-16
    154 lla-miR-16 1713 lla-mir-16
    155 sla-miR-16 1714 sla-mir-16
    156 mml-miR-16 1715 mml-mir-16-1
    1716 mml-mir-16-2
    157 mne-miR-16 1717 mne-mir-16
    158 ggo-miR-16 1718 ggo-mir-16
    159 ppa-miR-16 1719 ppa-mir-16
    360 ptr-miR-16 1720 ptr-mir-16
    161 ppy-miR-16 1721 ppy-mir-16
    162 lca-miR-16 1722 lca-mir-16
    163 cfa-miR-16 1723 cfa-mir-16-1
    1724 cfa-mir-16-2
    164 mdo-miR-16 1725 mdo-mir-16
    165 gga-miR-16 1726 gga-mir-16-1
    1727 gga-mir-16-2
    166 xtr-miR-16a 1728 xtr-mir-16a
    167 xtr-miR-16b 1729 xtr-mir-16b
    168 bta-miR-16 1730 bta-mir-16
    169 dre-rniR-1Ea 1731 dre-mir-16a
    170 dre-miR-16b 1732 dre-mir-16b
    171 dre-miR-16c 1733 dre-mir-16c
    172 fru-miR-16 1734 fru-mir-16
    173 tni-miR-16 1735 tni-mir-16
    174 mmu-miR-195 1736 mmu-mir-195
    175 mo-miR-195 1737 mo-mir-195
    176 mml-miR-195 1738 mml-mir-195
    177 ggo-miR-195 1739 ggo-mir-195
    178 ppa-miR-195 1740 ppa-mir-195
    179 cfa-miR-195 1741 cfa-mir-195
    180 bta-miR-195 1742 bta-mir-195
    181 rno-miR-17-3p 1743 rno-mir- 17-1
    1744 rno-mir-17-2
    182 age-miR-17-3p 1745 age-mir-17
    183 lla-miR-17-3p 1746 lla-mir-17
    184 sla-miR-17-3p 1747 sla-mir-17
    185 mml-miR-17-3p 174.8 mml-mir-17
    186 mne-miR-17-3p 1749 mne-mir-17
    187 ggo-miR-17-3p 1750 ggo-mir-17
    188 ppa-miR-17-3p 1751 ppa-mir-17
    189 ptr-miR-17-3p 1752 ptr-mir-17
    190 ppy-miR-17-3p 1753 ppv-mir-17
    191 Ica-miR-17-3p 1754 lca-mir-17
    192 mdo-miR-17-3p 1755 mdo-mir-17
    193 gga-miR-17-3p 1756 gga-mir-17
    194 xtr-miR-17-3p 1757 xtr-mir-17
    195 bta-miR-17-3p 1758 bta-mir-17
    196 mmu-miR-18a 1759 mmu-mir-18a
    197 rno-miR-18a 1760 rno-mir-18a
    198 age-miR-18 1761 age-mir-18
    199 lla-miR-18 1762 lla-mir-18
    200 sla-miR-18 1763 sla-mir-18
  • [Table 3-5]
    sequence number miRNA name sequence number miRNA precursor name
    201 mml-miR-18 1764 mml-mir-18
    202 mne-miR-18 1765 mne-mir-18
    203 ggo-miR-18 1766 ggo-mir-18
    204 ppa-miR-18 1767 ppa-mir-18
    205 ptr-miR-18 1768 ptr-mir-18
    206 ppy-miR-18 1769 ppy-mir-18
    207 lca-miR-18 1770 lca-mir-18
    208 mdo-miR-18 1771 mdo-mir-18
    209 gga-miR-18a 1772 gga-mir-18a
    210 xtr-miR-18a 1773 xtr-mir-18a
    211 bta-miR-18a 1774 bta-mir-18a
    212 ssc-miR-18 1775 ssc-mir-18
    213 dre-miR-18a 1776 dre-mir-18a
    214 dre-miR-18c 1777 dre-mir-18c
    215 fru-miR-18 1778 fru-mir-18
    216 tni-miR-18 1779 tni-mir-18
    217 mmu-miR-18b 1780 mmu-mir-18b
    218 mmi-miR-18b 1781 mml-mir-18b
    219 gga-miR-18b 1782 gga-mir-18b
    220 xtr-miR-18b 1783 xtr-mir-18b
    221 bta-miR-18b 1784 bta-mir-18b
    222 dre-miR-18b 1785 dre-mir-18b
    223 mmu-miR-20b 1786 mmu-mir-20b
    224 mo-miR-20a 1787 mo-mir-20a
    225 age-miR-20 1788 age-mir-20
    226 lla-miR-20 1789 lla-mir-20
    227 sla-miR-20 1790 sla-mir-20
    228 mml-miR-20a 1791 mml-mir-20a
    229 mml-miR-20b 1792 mml-mir-20b
    230 mne-miR-20 1793 mne-mir-20
    231 ggo-miR-20 1794 ggo-mir-20
    232 ppa-miR-20 1195 ppa-mir-20
    233 ptr-miR-20 1796 ptr-mir-20
    234 ppy-miR-20 1797 ppy-mir-20
    235 lca-miR-20 1798 lca-mir-20
    236 cfa-miR-20 1799 cfa-mir-20
    237 mdo-miR-20 1800 mdo-mir-20
    238 gga-miR-20a 1801 gga-mir-20a
    239 gga-miR-20b 1802 gga-mir-20b
    240 xla-miR-20 1803 xla-mir-20
    241 xtr-miR-20a 1804 xtr-mir-20a
    242 xtr-miR-20b 1805 xtr-mir-20b
    243 bta-miR-20a 1806 bta-mir-20a
    244 bta-miR-20b 1807 bta-mir-20b
    245 ssc-miR-20 1808 ssc-mir-20
    246 dre-miR-20a 1809 dre-mir-20a
    247 dre-miR-20b 1810 dre-mir-20b
    248 fru-miR-20 1811 fru-mir-20
    249 tni-miR-20 1812 tni-mir-20
    250 mmu-miR-21 1813 mmu-mir-21
  • [Table 3-6]
    sequence number miRNA name sequence number miRNA precursor name
    251 mo-miR-21 1814 mo-mir-21
    252 cgr-miR-21 1815 cgr-mir-21
    253 age-miR-21 1816 aga-mir-21
    254 mml-miR-21 1817 mml-mir-21
    255 mne-miR-21 1818 mne-mir-21
    256 Mo-miR-21 1819 ggo-mir-21
    257 ppa-miR-21 1820 ppa-mir-21
    258 ptr-miR-21 1821 ptr-mir-21
    259 ppy-miR-21 1822 ppy-mir-21
    260 cfa-miR-21 1823 cfa-mir-21
    261 mdo-miR-21 1824 mdo-mir-21
    262 gga-miR-21 1825 gga-mir-21
    263 bta-miR-21 1826 bta-mir-21
    264 ssc-miR-21 1827 ssc-mir-21
    265 dre-miR-21 1828 dre-mir-21-1
    1829 dre-mir-21-2
    266 fru-miR-2 1830 fru-mir-21
    267 tni-miR-21 1831 tni-mir-21
    268 mmu-miR-24 1832 mmu-mir-24-1
    1833 mmu-mir-24-2
    269 mo-miR-24 1834 mo-mir-24-1
    1835 mo-mir-24-2
    270 mml-miR-24 1836 mml-mir-24-1
    1837 mml-mir-24-2
    271 mne-miR-24 1838 mne-mir-24-1
    1839 mne-mir-24-2
    272 ggo-miR-24 1840 ggo-mir-24
    273 ppa-miR-24 1841 ppa-mir-24-1
    1842 ppa-mir-24-2
    274 ptr-miR-24 1843 ptr-mir-24
    275 ppy-miR-24 1844 ppy-mir-24-1
    1845 ppy-mir-24-2
    276 cfa-miR-24 1846 cfa-mir-24-1
    1847 cfa-mir-24-2
    277 mdo-miR-24 1848 mdo-mir-24-1
    1849 mdo-mir-24-2
    278 gga-miR-24 1850 gga-mir-24
    279 xtr-miR-24b 1851 xtr-mir-24b
    280 bta-miR-24 1852 bta-mir-24
    281 ssc-miR-24 1853 ssc-mir-24
    282 dre-miR-24 1854 dre-mir-24-1
    1855 dre-mir-24-2
    1856 dre-mir-24-3
    1857 dre-mir-24-4
    283 fru-miR-24 1858 fru-mir-24-1
    1859 fru-mir-24-2
    284 tni-miR-24 1860 tni-mir-24-1
    1861 tni-mir-24-2
    285 mmu-miR-25 1862 mmu-mir-25
    286 mo-miR-25 1863 rno-mir-25
    287 lla-miR-25 1864 lla-mir-25
    288 mml-miR-25 1865 mml-mir-25
    289 mne-miR-25 1866 mne-mir-25
    290 ggo-miR-25 1867 ggo-mir-25
    291 ppa-miR-25 1868 ppa-mir-25
    292 ppa-míR-25 1869 ptr-mir-25
    293 ppy-miR-25 1870 ppy-mir-25
    294 cfa-miR-25 1871 cfa-mir-25
    295 mdo-miR-25 1872 mdo-mir-25
    296 xtr-miR-25 1873 xtr-mir-25-1
    1814 xtr-mir-25-2
    297 bta-miR-25 1875 bta-mir-25
    298 dre-miR-25 1876 dre-mir-25
    299 fru-miR-25 1877 fru-mir-25
    300 tni-miR-25 1878 tni-mir-25
  • [Table 3-7]
    sequence number miRNA name sequence number miRNA precursor name
    301 mmu-miR-32 1879 mmu-mir-32
    302 rno-miR-32 1880 rno-mir-32
    303 sla-miR-32 1881 sla-mir-32
    304 mml-miR-32 1882 mml-mir-32
    305 mne-miR-32 1883 mne-mir-32
    306 ggo-miR-32 1884 ggo-mir-32
    307 ppa-miR-32 1885 ppa-mir-32
    308 ptr-miR-32 1886 ptr-mir-32
    309 ppy-miR-32 1887 ppy-mir-32
    310 cfa-miR-32 1888 cfa-mir-32
    311 mdo-miR-32 1889 mdo-mir-32
    312 gga-miR-32 1890 gga-mir-32
    313 ssc-miR-32 1891 ssc-mir-32
    314 mmu-miR-26b 1892 mmu-mir-26b
    315 rno-miR-26a 1893 rno-mir-26a
    316 rno-miR-26b 1894 rno-mir-26b
    317 lla-miR-26a 1895 lla-mir-26a
    318 mml-miR-26a 1896 mml-mir-26a-1
    1897 mml-mir-26a-2
    319 mml-miR-26b 1898 mml-mir-26b
    320 mne-miR-26a 1899 mne-mir-26a
    321 ggo-miR-26a 1900 ggo-mir-26a
    322 ppa-miR-26a 1901 ppa-mir-26a
    323 ptr-miR-26a 1902 ptr-mir-26a
    324 ppy-miR-26a 1903 ppy-mir-26a
    325 cfa-miR-26a 1904 cfa-mir-26a-1
    1905 cfa-mir-26a-2
    326 cfa-miR-26b 1906 cfa-mir-26b
    327 gga-miR-26a 1907 gga-mir-26a
    328 xtr-miR-26 1908 xtr-mir-26-1
    1909 xtr-mir-26-2
    329 bta-miR-26a 1910 bta-mir-26a
    330 bta-miR-26b 1911 bta-mir-26b
    331 ssc-miR-26a 1912 ssc-mir-26a
    332 dre-miR-26a 1913 dre-mir-26a-1
    1914 dre-mir-26a-2
    1915 dre-mir-26a-3
    333 dre-miR-26b 1916 dre-mir-26b
    334 fru-miR-26 1917 fru-mir-26
    335 tni-miR-26 1918 tni-mir-26
    336 mo-miR-30d* 1919 mo-mir-30d
    337 mo-miR-30e* 1920 mo-mir-30e
    338 mml-miR-30a-3p 1921 mml-mir-30a
    339 ggo-miR-30a-3p 1922 ggo-mir-30a
    340 ppa-miR-30a-3p 1923 ppa-mir-30a
    341 ptr-miR-30a-3p 1924 ptr-mir-30a
    342 ppy-miR-30a-3p 1925 ppy-mir-30a
    343 cfa-miR-30e 1926 cfa-mir-30e
    344 gga-miR-30a-3p 1927 gga-mir-30a
    345 xtr-miR-30a-3p 1928 xtr-mir-30a
    346 dre-miR-30e* 1929 dre-mir-30e-2
    347 mmu-miR-34a 1930 mmu-mir-34a
    348 mmu-miR-34b-5p 1931 mmu-mir-34b
    349 mo-miR-34a 1932 mn-mir-34a
    350 mo-miR-34c 1933 mo-mir-34c
  • [Table 3-8]
    sequence number miRNA name sequence number miRNA precursor name
    351 age-miR-34a 1934 age-mir-34a
    352 lla-miR-34a 1935 lla-mir-34a
    353 sla-miR-34a 1936 sla-mir-34a
    354 mml-miR-34a 1937 mml-mir-34a
    355 mml-miR-34c-5p 1938 mml-mir-34c
    356 mne-miR-34a 1939 mne-mir-34a
    357 ggo-miR-34a 1940 ggo-mir-34a
    358 ppa-miR-34a 1941 ppa-mir-34a
    359 ptr-miR-34a 1942 pfr-mir-34a
    360 ppy-miR-34a 1943 ppy-mir-34a
    361 cfa-miR-34a 1944 cfa-mir-34a
    362 cfa-miR-34c 1945 cfa-mir-34c
    363 mdo-miR-34a 1946 mdo-mir-34a
    364 gga-miR-34a 1947 gga-mir-34a
    365 gga-miR-34c 1948 gga-mir-34c
    366 xtr-miR-34a 1949 xtr-mir-34a
    367 bta-miR-34a 1950 bta-mir-34a
    368 bta-miR-34b 1951 bta-mir-34b
    369 bta-miR-34c 1952 bta-mir-34c
    370 dre-miR-34 1953 dre-mir-34
    371 dre-miR-34c 1954 dre-mir-34c
    372 mmu-miR-449a 1955 mmu-mir-449a
    373 mmu-miR-449c 1956 mmu-mir-449c
    374 mo-miR-449a 1957 ma-mir-449a
    375 mml-miR-449a 1958 mml-mir-449a
    376 cfa-miR-449 1959 cfa-mir-449
    377 mdo-miR-449 1960 mdo-mir-449
    378 gga-miR-449 1961 gga-mir-449
    379 xtr-miR-449 1962 xtr-mir-449
    380 mmu-miR-449b 1963 mmu-mir-449b
    381 mml-miR-449b 1964 mml-mir-449b
    382 mmu-miR-107 1965 mmu-mir-107
    383 mo-miR-107 1966 mo-mir-107
    384 lla-miR-107 1967 lla-mir-107
    385 mml-miR-107 1968 mml-mir-107
    386 mne-miR-107 1969 mne-mir-107
    387 ggo-miR-107 1970 ggo-mir-107
    388 ppa-miR-107 1971 ppa-mir-107
    389 ptr-miR-107 1972 ptr-mir-107
    390 ppy-miR-107 1973 ppy-mir-107
    391 cfa-miR-107 1974 cfa-mir-107
    392 mdo-miR-107 1975 mdo-mir-107
    393 gga-miR-107 1976 gga-mir-107
    394 xtr-miR-107 1977 xtr-mir-107
    395 bta-miR-107 1978 bta-mir-107
    396 ssc-miR-107 1979 ssc-mir- 07
    397 dre-miR-107 1980 dre-mir-107
    398 fru-miR-107 1981 fru-mir-107
    399 tni-miR-107 1982 tni-mir-107
    400 ptr-miR-140 1983 pfr-mir-140
  • [Table 3-9]
    sequence number miRNA name sequence number miRNA precursor name
    401 gga-miR-140 1984 gga-mir-140
    402 xtr-miR-140 1985 xtr-mir-140
    403 ssc-miR-140 1986 ssc-mir-140
    404 mmu-miR-148b 1987 mmu-mir-148b
    405 mml-miR-148a 1988 mml-mir-148a
    406 mml-miR-148b 1989 mml-mir-148b
    407 cfa-miR-148a 1990 cfa-mir-148a
    408 cfa-miR-148b 1991 cfa-mir-148b
    409 gga-miR-148a 1992 gga-mir-148a
    410 xtr-miR-148a 1993 xtr-mir-148a
    411 xtr-miR-448b 1994 xtr-mir-148b
    412 bta-miR-148a 1995 bta-mir-148a
    413 bta-miR-148b 1996 bta-mir-148b
    414 ssc-miR-148a 1997 ssc-mir-148a
    415 dre-miR-148 1998 dre-mir-148
    416 fru-miR-148 1999 fru-mir-148
    417 tni-miR-148 2000 tni-mir-148
    418 mmu-miR-190 2001 mmu-mir-190
    419 mo-miR-190 2002 ma-mir-190
    420 mo-miR-190b 2003 mo-mir-190b
    421 mml-miR-190a 2004 mml-mir-190a
    422 mml-miR-190b 2005 mml-mir-190b
    423 ggo-miR-190 2006 ggo-mir-190
    424 ppa-miR-190 2007 ppa-mir-190
    425 ptr-miR-190 2008 ptr-mir-190
    426 gga-miR-190 2009 gga-mir-190
    427 dre-miR-190 2010 dre-mir-190
    428 dre-miR-190b 2011 dre-mir-190b
    429 fru-miR-190 2012 fru-mir-190
    430 tni-miR-190 2013 tni-mir-190
    431 lla-miR-199a 2014 lla-mir-199a
    432 sla-miR-199a 2015 sla-tnir-199a
    433 mml-miR-199a 2016 mml-mir-199a-1
    2017 mml-mir-199a-2
    434 mne-miR-189a 2018 mne-mir-199a
    435 mo-miR-199a 2019 ggo-mir-199a
    436 ppa-miR-199a 2020 ppa-mir-199a
    437 ptr-mir-199a 2021 ptr-mir-199a
    438 ppy-miR-199a 2022 ppy-mir-199a
    439 xtr-miR-199a 2023 xtr-mir-199a
    440 dre-miR-199 2024 dre-mir-199-1
    2025 dre-mir-199-2
    2026 dre-mir-199-3
    441 fru-miR-199 2027 fru-mir-199-1
    2028 fru-mir-199-2
    2029 fru-mir-199-3
    442 tni-miR-199 2030 tni-mir-199-1
    2031 tni-mir-199-2
    2032 tni-mir-199-3
    443 mdo-miR-199b 2033 mdo-mir-199b
    444 xtr-miR-199b 2034 xtr-mir-199b
    445 bta-miR-199b 2035 bta-mir-199b
    446 gga-miR-202* 2036 gna-mir-202
    447 xtr-miR-202* 2037 xtr-mir-202-1
    2038 xtr-mir-202-2
    448 mmu-miR-208b 2039 mmu-mir-208b
    449 mo-miR-208 2040 mo-mir-208
    450 mml-miR-208a 2041 mml-mir-208a
  • [Table 3-10]
    sequence number miRNA name sequence number miRNA precursor name
    451 mml-miR-208b 2042 mml-mir-208b
    452 mdo-miR-208 2043 mdo-mir-208
    453 xtr-miR-208 2044 xtr-mir-208
    454 mmu-miR-210 2045 mmu-mir-210
    455 mmu-miR-210 2046 mo-mir-210
    456 mml-miR-210 2047 mml-mir-210
    457 xtr-miR-210 2048 xtr-mir-210
    458 dre-miR-210 2049 dre-mir-210
    459 fru-miR-210 2050 fru-mir-210
    460 tni-miR-210 2051 tni-mir-210
    461 mnu-miR-21 2052 mmu-mir-211
    462 mo-niR-211 2053 mo-mir-211
    463 mml-miR-211 2054 mml-mir-211
    464 mne-miR-211 2055 mne-mir-211
    465 ppy-miR-211 2056 ppy-mir-211
    466 gga-miR-211 2057 gga-mir-211
    467 mmu-miR-214 2058 mmu-mir-214
    468 mo-miR-214 2059 ma-mir-214
    469 age-miR-214 2060 age-mir-214
    470 sla-miR-214 2061 sla-mir-214
    471 mml-miR-214 2062 mml-mir-214
    472 mnr-miR-214 2063 mne-mir-214
    473 ggo-miR-214 2064 ggo-mir-214
    474 ppa-miR-214 2085 ppa-mir-214
    475 ptr-miR-214 2066 ptr-mir-214
    476 ppy-miR-214 2067 ppy-mir-214
    477 mdo-miR-214 2068 mdo-mir-214
    478 xtr-miR-214 2069 xtr-mir-214
    479 bta-miR-214 2070 bta-mir-214
    480 ssc-miR-214 2071 ssc-mir-214
    481 dre-miR-214 2072 dre-mir-214
    482 fru-miR-214 2073 fru-mir-214
    483 tni-miR-214 2074 tni-mir-214
    484 mmu-miR-218 2075 mmu-mir-218-1
    2076 mmu-mir-218-2
    485 mo-miR-218 2077 mo-mir-218-1
    2078 mo-mir-218-2
    486 age-miR-218 2079 age-mir-218
    487 lla-miR-218 2080 lla-mir-218-1
    2081 lla-mir-218-2
    488 sla-miR-218 2082 sla-mir-218-1
    2083 sla-mir-218-2
    489 mml-miR-218 2084 mml-mir-218-1
    2085 mml-mir-218-2
    490 mne-miR-218 2086 mne-mir-218-1
    2087 mne-mir-218-2
    491 ggo-miR-218 2088 ggo-mir-218
    492 ppa-miR-218 2089 ppa-mir-218-1
    2090 ppa-mir-218-2
    493 ptr-miR-218 2091 ptr-mir-218-1
    2092 ptr-mir-218-2
    494 ppy-miR-218 2093 ppy-mir-218-1
    2094 ppy-mir-218-2
    495 lca-miR-218 2095 lca-mir-218
    496 cfa-miR-218 2096 cfa-mir-218-1
    2097 cfa-mir-218-2
    497 mdo-miR-218 2098 mdo-mir-218
    498 gga-miR-218 2099 gga-mir-218-1
    2100 gga-mir-218-2
    2101 gga-mir-218-3
    499 xtr-miR-218 2102 xtr-mir-218-1
    2103 xtr-mir-218-2
    500 bta-miR-218 2104 bta-mir-218
  • [Table 3-11]
    sequence number miRHA name sequence number miRNA precursor name
    501 dre-miR-218b 2105 dre-mir-218b
    502 fru-miR-218a 2106 fru-mir-218a-1
    2107 fru-mir-218a-2
    503 fru-miR-218b 2108 fru-mir-218b
    504 tni-miR-218a 2109 tni-mir-218a-1
    2100 tni-mir-218a-1
    505 tni-miR-218b 2111 tni-mir-218b
    506 mml-miR-299-5p 2112 mml-mir-299
    507 mml-miR-325 2113 mml-mir-325
    508 ssc-miR-325 2114 ssc-mir-325
    509 mmu-miR-328 2115 mmu-mir-328
    510 mo-miR-328 2116 mo-mir-328
    511 cfa-miR-328 2117 cfa-mir-328
    512 mmu-miR-329 2118 mmu-mir-329
    513 mo-miR-329 2119 mo-mir-329
    514 mml-miR-329 2120 mnl-mir-329-1
    2121 mml-mir-329-2
    515 mo-miR-338 2122 mo-mir-338
    516 cfa-miR-338 2123 cfa-mir-838
    517 mdo-miR-338 2124 mdo-mir-338
    518 xtr-miR-338 2125 xtr-mir-338-1
    2126 xtr-mir-338-2
    519 dre-miR-338 2127 dre-mir-338-1
    2128 dre-mir-338-2
    2129 dre-mir-338-3
    2130 dre-mir-338-4
    520 fru-miR-338 2131 fru-mir-338
    521 tni-miR-338 2132 tni-mir-338
    522 xtr-miR-425-5p 2133 xtr-mir-425
    523 mmu-miR-484 2134 mmu-mir--484
    524 mo-miR-484 2135 mo-mir-484
    525 mml-miR-484 2136 mml-mir-484
    526 bta-miR-484 2137 bta-mir-484
    527 mml-miR-485-5p 2138 mml-mir-485
    528 mmu-miR-486 2139 mmu-mir-486
    529 mml-miR-520b 2140 mml-mir-520b
    530 mml-miR-520c 2141 mml-mir-520c
    531 mml-miR-520e 2142 mml-mir-520e
    532 mml-miR-520f 2143 mml-mir-520f
    533 mml-miR-520h 2144 mml-mir-520h
    534 mml-miR-522 2145 mml-mir-522
    535 mml-miR-573 2146 mml-mir-573
    536 mml-miR-650b 2147 mml-mir-650b
    537 mml-miR-650c 2148 mml-mir-650c
    538 mml-miR-660 2149 mml-mir-660
    539 cfa-miR-660 2150 cfa-mir-660
    540 bta-miR-660 2151 bta-mir-660
    541 mmu-miR-668 2152 mmu-mir-668
    542 mml-miR-668 2153 mml-mir-668
    543 mml-miR-675 2154 mm-mir-675
    544 mml-miR-765 2155 mml-mir-765
    545 mmu-miR-194 2156 mmu-mir-194-1
    2157 mmu-mir-194-2
    546 mo-miR-194 2158 mo-mir-194-1
    2159 mo-mir-194-2
    547 age-miR-194 2160 age-mir-194
    548 mml-miR-194 2161 mml-mir-194-1
    2162 mml-mir-194-2
    549 mme-miR-194 2163 mne-mir-194
    550 ggo-miR-194 2164 ggo-mir-194
  • [Table 3-12]
    sequence miRNA name sequence miRNA precusor name
    551 ptr-miR-194 2165 ptr-mir-194
    552 ppy-miR-194 2166 ppy-mir-194
    553 cfa-miR-194 2167 cfa-mir-194
    554 gga-miR-194 2168 gga-mir-194
    555 xtr-miR-194 2169 xtr-mir-194-1
    2170 xtr-mir-194-2
    556 dre-miR-194b 2171 dre-mir-194b
    557 fru-miR-194 2172 fru-mir-194
    558 tni-miR-194 2173 tri-mir-194
    559 cfa-miR-500 2174 cfa-mir-500
    560 mmu-miR-197 2175 mmu--mir-197
    561 age-miR-197 2176 age-mir-197
    562 mml-miR-197 2177 mml-mir-197
    563 mme-miR-197 2178 mne-mir-197
    564 ppa-miR-197 2179 ppa-mir-197
    565 ptr-miR-197 2180 ptr-mir-197
    566 ppy-miR-197 2181 ppy-mir-197
    567 cfa-miR-197 2182 cfa-mir-197
    568 mmu-miR-221 2183 mmu-mir-221
    569 mo-miR-221 2184 mo-mir-221
    570 mml-miR-221 2185 mml-mir-221
    571 ggo-miR-221 2186 ggo-mir-221
    572 ppa-miR-221 2187 ppa-mir-221
    573 ppy-miR-221 2188 ppy-mir-221
    574 cfa-miR-221 2189 cfa-mir-221
    575 mdo-miR-221 2190 mdo-mir-221
    576 gga-miR-221 2191 gga-mir-221
    577 xtr-miR-221 2192 xtr-mir-221
    578 bta-miR-221 2193 bta-mir-221
    579 dre-mir-221 2194 dre-mir-221
    580 fru-miR-221 2195 fru-mir-221
    581 tni-miR-221 2196 tni-mir-22
    582 mmu-let-7a 2197 mmu-let-7a-1
    2198 mmu-let-7a-2
    583 mmu-let-7b 2199 mmu-let-7b
    584 mmu-let-7c 2200 mmu-let-7c-1
    2201 mmu-let-7c-2
    585 mmu-let-7d 2202 mmu-let-7d
    586 mmu-let-7e 2203 mmu-let-7e
    587 mmu-let-7f 2204 mmu-let-7f-1
    2205 mmu-let-7f-2
    588 mmu-let-7i 2206 mmu-let-7i
    589 rno-let-7a 2207 rno-let-7a-1
    2208 rno-let-7a-2
    590 rno-let-7b 2209 rno-let-7b
    591 rno-let-7c 2210 rno-let-7c-1
    2211 rno-let-7c-2
    592 rno-let-7d 2212 rno-let-7d
    593 rno-let-7e 2213 rno-let-7e
    594 rno-let-7f 2214 rno-let-7f-1
    2215 rno-let-7f-2
    595 rno-let-7i 2216 rno-let-7i
    586 nml-let-7a 2217 mml-let-7a-1
    2218 mml-let-7a-2
    2219 mml-let-7a-3
    597 mml-let-7b 2220 mm-let-7b
    598 mml-llet-7c 2221 mml-let-7c
    599 mml-let-7d 2222 mml-let-7d
    600 mml-let-7e 2223 mml-let-7e
  • [Table 3-13]
    sequence number miRMA name sequence number miRNA precursor name
    601 mml-let-7f 2224 mm-let-7f-1
    2225 mml-let-7f-2
    602 mml-lest-7g 2226 mml-let-7g
    603 mm-let-7i 2227 mml-let-7i
    604 cfa-let-7a 2228 cfa-let-7a
    605 cfa-let-7c 2229 cfa-let-7c
    606 cfa-let-7e 2230 cfa-let-7e
    607 cfa-let-7f 2231 cfa-let-7f
    608 cfa-let-7g 2232 cfa-let-7g
    609 cfa-let-7j 2233 cfa-let-7j
    610 mdo-let-7a 2234 mdo-let-7a-1
    2235 mdo-let-7a-2
    2236 mdo-let-7a-3
    611 mdo-let-7b 2237 mdo-let-76
    612 mdo-let-7d 2238 mdo-let-7d
    613 mdo-let-7f 2239 mdo-let-7f-1
    2240 mdo-let-7f-2
    614 mdo-let-7g 2241 mdo-let-7g
    615 mdo-let-7i 2242 mdo-let-7i
    616 gga-let-7a 2243 gga-let-7a-1
    2244 gga-let-7a-2
    2245 gga-let-7a-3
    617 gga-let-7b 2246 gga-let-7b
    618 gga-let-7c 2247 gga-let-7c
    619 gga-let-7d 2248 gga-let-7d
    620 gga-let-7f 2249 gga-let-7f
    621 gga-let-7g 2250 gga-let-7g
    622 gga-let-7i 2251 gga-let-7i
    623 gga-let-7i 2252 gga-let-7j
    624 gga-let-7k 2253 gga-let-7k
    625 xtr-let-7a 2254 xtr-let-7a
    626 xtr-let-7b 2255 xtr-let-7b
    627 xtr-let-7c 2256 xtr-let-7c
    628 xtr-let-7e 2257 xtr-let-7e-1
    2258 xtr-let-7e-2
    629 xtr-let-7f 2259 xtr-let-7f
    630 xtr-let-7g 2260 xtr-let-7g
    631 xtr-let-7i 2261 xtr-let-7i
    632 bta-let-7a 2262 bta-let-7a-1
    2263 bta-let-7a-2
    2264 bta-let-7a-3
    633 bta-let-7b 2265 bta-let-7b
    634 bta-let-7c 2266 bta-let-7c
    635 bta-let-7d 2267 bta-let-7d
    636 bta-let-7e 2268 bta-let-7e
    637 bta-let-7f 2269 bta-let-7f-1
    2270 bta-let-7f-2
    638 bta-let-7g 2271 bta-let-7g
    639 bta-let-7i 2272 bta-let-7i
    640 ssc-let-7c 2273 ssc-let-7c
    641 ssc-let-7f 2274 ssc-let-7f
    642 ssc-let-7i 2275 ssc-let-7i
    643 dre-let-7a 2276 dre-let-7a-1
    2277 dre-let-7a-2
    2278 dre-let-7a-3
    2279 dre-let-7a-4
    2280 dre-let-7a-5
    2281 dre-let-7a-6
    644 dre-let-7b 2282 dre-let-7b
    645 dre-let-7c 2283 dra-let-7c-1
    2284 dre-lat-7c-2
    646 dre-let-7d 2285 dre-let-7d-1
    2286 dre-let-7d-2
    647 dre-let-7e 2287 dre-let-7e
    648 dre-let-7f 2288 dre-let-7f
    649 dre-let-7g 2289 dre-let-7g-1
    2290 dre-let-7g-2
    650 dre-let-7h 2291 dra-let-7h
  • [Table 3-14]
    sequence number miRNA name sequence number miRNA precursor name
    651 dre-let-7i 2292 dre-let-7i
    652 dre-let-7j 2293 dre-let-7j
    653 fru-let-7a 2294 fru-let-7a-1
    2295 fru-let-7a-2
    2296 fru-let-7a-3
    654 fru-let-7b 2297 fru-let-7b
    655 fru-let-7d 2298 fru-let-7d
    656 fru-let-7e 2299 fru-let-7e
    657 fru-let-7g 2300 fru-let-7g
    658 fru-let-7h 2301 fru-let-7h
    659 fru-let-7i 2302 fru-lat-7i
    660 fru-1et-7j 2303 fru-let-7j
    661 tni-let-7a 2304 tni-let-7a-1
    2305 tni-let-7a-2
    2306 tni-let-7a-3
    662 tni-let-7b 2307 tni-let-7b
    663 tni-let-7d 2308 tni-let-7d
    664 tni-let-7e 2309 tni-let-7e
    665 tni-let-7g 2310 tni-let-7g
    666 tni-let-7h 2311 tni-let-7h
    667 tni-let-7i 2312 tni-let-7i
    668 tni-let-7j 2313 tni-let-7j
    669 mmu-miR-1 2314 mmu-mir-1-1
    2315 mmu-mir-1-2
    2316 mm-mir-1-2-as
    670 mo-miR-1 2317 mo-mir-1
    671 mml-miR-1 2318 mml-mir-1-1
    2319 mml-mir-1-2
    672 ppa-miR-1 2320 ppa-mir-1
    673 cfa-miR-1 2321 cfa-mir-1-1
    2322 cfa-mir-1-2
    674 mdo-miR-1 2323 mdo-mir-1
    675 gga-miR-1a 2324 gga-mir-1a-1
    2325 gga-mir-1a-2
    676 gga-miR-1b 2326 gga-mir-1b
    677 xtr-miR-1b 2327 xtr-mir-1b
    678 dre-miR-1 2328 dre-mir-1-1
    2329 dre-mir-1-2
    679 fru-miR-1 2330 fru-mir1
    680 tri-miR-1 2331 tni-mir-1
    681 mmu-miR-206 2332 mmu-mir-206
    682 mo-miR-206 2333 mo-mir-206
    683 mml-miR-206 2334 mml-mir-206
    684 mne-miR-206 2335 mne-mir-206
    685 ppy-miR-206 2336 ppy-mir-206
    686 cfa-miR-206 2337 cfa-mir-206
    687 mdo-miR-206 2338 mdo-mir-206
    688 gga-miR-206 2339 gga-mir-206
    689 xtr-miR-206 2340 xtr-mir-206
    690 dre-miR-206 2341 dre-mir206-1
    2342 dre-mir-206-2
    691 mmu-miR-9 2343 mmu-mir-9-1
    2344 mmu-mir-9-2
    2345 mmu-mir-9-3
    692 mo-miR-9 2346 mo-mir-9-1
    2347 mo-mir-9-2
    2348 mo-mir-9-3
    693 age-miR-9 2349 age-mir-9
    694 lla-miR-9 2350 lla-mir-9
    695 mml-miR-9 2351 mml-mir-9-1
    2352 mml-mir-9-2
    2353 mml-mir-9-3
    696 mne-miR-9 2354 mne-mir-9
    697 ggo-miR-9 2355 ggo-mir-9
    698 ptr-miR-9 2356 ptr-mir-9
    699 cfa-miR-9 2357 cfa-mir-9-1
    2358 cfa-mir-9-2
    2359 cfa-mir9-3
    700 mdo-miR-9 2360 mdo-mir-9-1
    2361 mdo-mir-9-2
  • [Table 3-15]
    sequence number miRNA name sequence number miRNA precursor name
    701 gga-miR-9 2362 gga-mir-9-1
    2363 gga-mir-9-2
    702 xtr-miR-9 2364 xtr-mir-9-3
    703 xtr-miR-9b 2365 xtr-mir-3b
    704 dre-miR-9 2366 dre-mir-9-1
    2367 dre-mir-9-2
    2368 dre-mir-9-3
    2369 dre-mir-9-4
    2370 dre-mir-9-5
    2371 dre-mir-9-6
    2372 dre-mir-9-7
    705 fru-miR-9 2373 fru-mir-9-1
    2374 fru-mir-9-2
    2375 fru-mir-9-3
    2376 fru-mir-9-4
    706 tni-miR-9 2377 tni-mir-9-1
    2378 tn-mir-9-2
    2379 tni-mir-9-3
    2380 tni-mir-9-4
    707 mmu-miR-23b 2381 mmu-mir-23b
    708 mo-miR-23a 2382 mo-mir-23a
    709 mo-miR-23b 2383 mo-mir-23b
    710 age-miR-23a 2384 age-mir-23a
    711 sla-miR-23a 2385 sla-mir-23a
    712 sla-miR-23a 2386 mml-mir-23a
    713 mml-miR-23b 2387 mml-mir-23b
    714 mme-miR-23a 2388 mme-mir-23a
    715 ggo-miR-23a 2389 ggo-mir-23a
    716 ppa-miR-23a 2390 ppa-mir-23a
    717 ppa-miR-28b 2391 ppa-mir-23b
    718 ptr-miR-23a 2392 ptr-mir-23a
    719 ptr-miR-23b 2393 ptr-mir-23b
    720 ppy-miR-23a 2394 ppy-mir-23a
    721 ppy-miR-23b 2395 ppy-mir-23b
    722 lca-miR-23a 2396 lca-mir-23a
    723 cfa-miR-23a 2397 cfa-mir-23a
    724 cfa-miR-23b 2398 cfa-mir-23b
    725 mdo-miR-23a 2399 mdo-mir-23a
    726 mdo-miR-23b 2400 mdo-mir-23b
    727 gga-miR-23b 2401 gga-mir-23b
    728 xtr-miR-23 2402 xtr-mir-23a-1
    2403 xtr-mir-23a-2
    729 xtr-miR-23b 2404 xtr-mir-23b
    730 bta-miR-23a 2405 bta-mir-23a
    731 bta-miR-23b 2406 bta-mir-23b
    732 ssc-miR-23a 2407 ssc-mir-23a
    733 dre-miR-23a 2408 dre-mir-23a-1
    2409 dre-mir-23a-2
    2410 dre-mir-23a-3
    734 dre-miR-23b 2411 dre-mir-23b
    735 fru-miR-23a 2412 fru-mir-23a-1
    2413 fru-mir-23a-2
    2414 fru-mir-23a-3
    736 fru-miR-23b 2415 fru-mir-23b
    737 tni-miR-23a 2416 tri-mir-23a-1
    2417 tni-mir-23a-2
    2418 tni-mir-23a-3
    738 tni-miR-23b 2419 tri-mir-23b
    739 mmu-miR-27b 2420 mmu-mir-27b
    740 mo-miR-27e 2421 mo-mir-27a
    741 mo-miR-27b 2422 mo-mir-27b
    742 mo-miR-27a 2423 age-mir-27a
    743 sla-miR-27a 2424 sla-mir-27a
    744 mml-miR-27a 2425 mml-mir-27a
    745 mml-miR-27b 2426 mml-mir-27b
    746 mme-miR-27a 2427 mne-mir-27a
    747 ggo-miR-27a 2428 ggo-mir-27a
    748 ppa-miR-27a 2429 ppa-mir-27a
    749 ptr-miR-27a 2430 ptr-mir-27a
    750 ppy-miR-27a 2431 ppy-mir-27a
  • [Table 3-16]
    sequence number miRNA name sequence number miRNA precursor name
    751 lca-miR-27a 2432 lca-mir-27a
    752 cfa-miR-27a 2433 cfa-mir-27a
    753 cfa-miR-27b 2434 cfa-mir-21b
    754 mdo-miR-27a 2435 mdo-mir-27a
    755 mdo-miR-27b 2436 mdo-mir-27b
    756 gga-miR-27b 2437 gga-mir-27b
    757 xtr-miR-27a 2438 xtr-mir-27a
    758 xtr-miR-27b 2439 xtr-mir-27b
    759 xtr-miR-27c 2440 xtr-mir-27c-1
    2441 xtr-mir-27c-2
    760 bta-miR-27a 2442 bta-mir-27a
    761 bta-miR-27b 2443 bta-mir-27b
    762 ssc-miR-27a 2444 ssc-mir-27a
    763 dre-miR-27a 2445 dre-mir-27a
    764 dre-miR-27b 2446 dre-mir-27b
    765 dre-miR-27c 2447 dre-mir-27c
    766 dre-miR-27d 2448 dre-mir-27d
    761 dre-miR-27e 2449 dre-mir-27e
    768 fru-miR-27b 2450 fru-mir-27b
    769 fru-miR-27c 2451 fru-mir-27c
    770 fru-miR-27e 2452 fru-mir-27e
    771 tni-miR-27b 2453 tni-mir-27b
    772 tni-miR-27c 2454 tni-mir-27c
    773 tni-miR-27e 2455 tni-mir-27e
    774 mmu-miR-28 2456 mmu-mir-28
    775 mo-miR-28 2457 mo-mir-28
    776 age-miR-28 2458 age-mir-28
    777 lla-miR-28 2459 lla-mir-28
    778 sla-miR-28 2460 sla-mir-28
    779 mml-miR-28 2461 mml-mir-28
    780 mne-miR-28 2462 mne-mir-28
    781 ggo-miR-28 2463 ggo-mir-28
    782 ppa-miR-28 2464 ppa-mir-28
    783 ptr-miR-28 2465 ptr-mir-28
    784 ppy-miR-28 2466 ppy-mir-28
    785 ssc-miR-28 2467 ssc-mir-28
    786 mml-miR-31 2468 mml-mir-31
    787 mne-miR-31 2469 mne-mir-31
    788 ggo-miR-31 2470 ggo-mir-31
    789 ppa-miR-31 2471 ppa-mir-31
    790 ptr-miR-31 2472 ptr-mir-31
    791 ppy-miR-31 2473 ppy-mir-31
    792 dre-miR-31 2474 dre-mir-31
    793 mo-miR-33 2475 mo-mir-33
    794 mml-miR-33a 2476 mml-mir-33a
    795 mml-miR-33b 2477 mml-mir-33b
    796 mne-miR-33 2478 mne-mir-33
    797 ggo-miR-33 2479 ggo-mir-33
    798 ppa-miR-33 2480 ppa-mir-33
    799 ptr-miR-33 2481 ptr-mir-33
    800 ppy-miR-33 2482 ppy-mir-33
  • [Table 3-17]
    sequence number miRNA name sequence number miRNA precursor name
    801 cfa-miR-33 2483 cfa-mir-33
    802 gga-miR-33 2484 gga-mir-33
    803 xtr-miR-33a 2485 xtr-mir-33a
    804 xtr-miR-33b 2486 xtr-mir-33b
    805 mmu-miR-96 2487 mmu-mir-96
    806 mo-miR-96 2488 mo-mir-96
    807 sla-miR-96 2489 sla-mir-96
    808 mml-miR-96 2490 mml-mir-96
    809 mne-miR-96 2491 mne-mir-86
    810 ggo-miR-96 2492 ggo-mir-96
    811 ppa-miR-96 2493 ppa-mir-96
    812 ptr-miR-96 2494 ptr-mir-96
    813 mdo-miR-96 2495 mdo-mir-96
    814 xtr-miR-96 2496 xtr-mir-96
    815 dre-miR-96 2497 dre-mir-96
    816 fru-miR-96 2498 fru-mir-96
    817 tni-miR-96 2499 tni-mir-96
    818 mmu-miR-100 2500 mmu-mir-100
    819 mo-miR-100 2501 mo-mir-100
    820 age-miR-100 2502 age-mir-100
    821 lla-miR-100 2503 lla-mir-100
    822 sla-miR-100 2504 sla-mir-100
    823 mml-miR-100 2505 mml-mir-100
    824 ggo-miR-100 2506 ggo-mir-100
    825 ppa-miR-100 2507 ppa-nir-100
    826 ptr-miR-10 2508 ptr-mir-100
    827 ppy-miR-100 2509 ppy-mir-100
    828 mdo-miR-100 2510 mdo-mir-100
    829 gga-miR-100 2511 gga-mir-100
    830 xtr-miR-100 2512 xtr-mir-100
    831 dre-miR-100 2513 dre-mir-100-1
    2514 dre-mir-100-2
    832 fru-miR-100 2515 fru-mir-100
    833 tni-miR-100 2516 tri-mir-100
    834 mmu-miR-106a 2517 mmu-mir-106a
    835 rno-miR-106b 2518 rno-mir-106b
    836 age-miR-106a 2519 age-mir-106a
    837 age-miR-106b 2520 age-mir-106b
    838 lla-miR-106b 2521 lla-mir-106b
    839 sla-miR-106a 2522 sla-mir-106a
    840 sla-miR-106b 2523 sla-mir-106b
    841 mml-miR-106a 2524 mml-mir-106a
    842 mml-miR-106b 2525 mml-mir-106b
    843 mne-miR-106a 2526 mne-mir-106a
    844 mne-miR-106b 2527 mne-mir-106b
    845 ggo-miR-106a 2528 ggo-mir-106a
    846 ggo-miR-106b 2529 ggo-mir-106b
    847 ppa-miR-106a 2530 ppa-mir-106a
    848 ppa-miR-106b 2531 ppa-mir-106b
    849 ptr-miR-106a 2532 ptr-mir-106a
    850 ptr-miR-106b 2533 ptr-mir-106b
  • [Table 3-18]
    sequence number miRNA name sequence number miRNA precursor name
    851 ppy-miR-106a 2534 ppy-mir-106a
    852 ppy-miR-106b 2535 ppy-mir-106b
    853 cfa-miR-106b 2536 cfa-mir-106b
    854 gga-miR-106 2537 gga-mir-106
    855 xtr-miR-106 2538 xtr-mir-106
    856 bta-miR-106 2539 bta-mir-106
    857 ssc-miR-106a 2540 ssc-mir-106a
    858 rno-miR-126* 2541 rno-mir-126
    859 gga-miR-126* 2542 gga-mir-126
    860 xtr-miR-126* 2543 xtr-mir-126
    861 bta-miR-126* 2544 bta-mir-126
    862 dre-miR-126* 2545 dre-mir-126
    863 mmu-miR-127 2546 mmu-mir-127
    864 rno-miR-127 2547 rno-mir-127
    865 age-miR-127 2548 age-mir-127
    866 lla-miR-127 2549 lla-mir-127
    867 sla-miR-127 2550 sla-mir-127
    868 mml-miR-127 2551 mml-mir-127
    869 mne-miR-127 2552 mne-mir-127
    870 ptr-miR-127 2553 ptr-mir-127
    871 ppy-miR-127 2554 ppy-mir-127
    872 cfa-miR-127 2555 cfa-mir-127
    873 bta-miR-127 2556 bta-mir-127
    874 rno-miR-128 2557 rno-mir-128-1
    2558 rno-mir-128-2
    875 age-miR-128 2559 age-mir-128
    876 sla-miR-128 2560 sla-mir-128
    877 mml-miR-128a 2561 mml-mir-128a
    878 mml-miR-128b 2562 mml-mir-128b
    879 ppa-miR-128 2563 ppa-mir-128
    880 ptr-miR-128 2564 ptr-mir-128
    881 ppy-miR-128 2565 ppy-mir-128
    882 882 cfa-miR-128 2566 cfa-mir-128-1
    2567 cfa-mir-128-2
    883 mdo-miR-128 2568 mdo-mir-128
    884 gga-miR-128 2569 gga-mir-128-1
    2570 gga-mir-128-2
    885 xtr-miR-128 2571 xtr-mir-128-1
    2572 xtr-mir-128-2
    886 bta-miR-128 2573 bta-mir-128
    887 ssc-miR-128 2574 ssc-mir-128
    888 dre-miR-128 2575 dre-mir-128-1
    2576 dre-mir-128-2
    889 fru-miR-128 2577 fru-mir-128-1
    2578 fru-mir-128-2
    890 tni-miR-128 2579 tni-mir-128-1
    2580 tni-mir-128-2
    891 rno-miR-129 2581 rno-mir-129-1
    2582 rno-mir-129-2
    892 cfa-miR-129 2583 cfa-mir-129-1
    2584 cfa-mir-129-2
    893 mdo-mir-129 2585 mdo-mir-129
    894 xtr-miR-129 2586 xtr-mir-129-1
    2587 xtr-mir-128-2
    895 dre-miR-129 2588 dre-mir-129-1
    2589 dre-mir-129-2
    896 fru-miR-129 2590 fru-mir-129-1
    2591 fru-mir-129-2
    897 tni-miR-129 2592 tni-mir-129-1
    2593 tni-mir-129-2
    898 age-miR-133a 2594 age-mir-133a
    899 lla-miR-133a 2595 lla-mir-133a
    900 sla-miR-133a 2596 sla-mir-133a
  • [Table 3-19]
    sequence number miRNA name sequence number miRNA precursor name
    901 mne-miR-133a 2597 mne-mir-133a
    902 ggo-miR-133a 2598 ggo-mir-133a
    903 ppa-miR-133a 2599 ppa-mir-133a
    904 ptr-miR-133a 2600 ptr-mir-133a
    905 mdo-miR-133a 2601 mdo-mir-133a
    906 gga-miR-133a 2602 gga-mir-133a-1
    2603 gga-mir-133a-2
    907 gga-miR-133c 2604 gga-mir-133c
    908 xla-miR-133a 2605 xla-mir-133a
    909 xtr-miR-133a 2606 xtr-mir-133a
    910 xtr-miR-133d 2607 xtr-mir-133d
    911 gga-miR-133b 2608 gga-mir-133b
    912 xtr-miR-133b 2609 xtr-mir-133b
    913 mmu-miR-135b 2610 mmu-mir-135b
    914 rno-miR-135a 2611 rno-mir-135a
    915 rno-miR-135b 2612 rno-mir-135b
    916 age-miR-135 2613 age-mir-135-1
    2614 age-mir-135-2
    917 lla-miR-135 2615 lla-mir-135-1
    2616 lla-mir-135-2
    918 mml-miR-135a 2617 mml-mir-135a-1
    2618 mml-mir-135a-2
    919 mml-miR-135b 2619 mml-mir-135b
    920 ggo-miR-135 2620 ggo-mir-135
    921 ppa-miR-135 2621 ppa-mir-135-1
    2622 ppa-mir-135-2
    922 ptr-miR-135 2623 ptr-mir-135
    923 ppy-miR-135 2624 ppy-mir-135
    924 mdo-miR-135a 2625 mdo-mir-135a
    925 mdo-miR-135b 2626 mdo-mir-135b
    926 gga-miR-135a 2627 gga-mir-135a-1
    2628 gga-mir-135a-2
    2629 gga-mir-135a-3
    927 xtr-miR-135 2630 xtr-mir-135-1
    2631 xtr-mir-135-2
    928 ssc-miR-135 2632 ssc-mir-135-1
    2633 ssc-mir-135-2
    929 dre-miR-135a 2634 dre-mir-135a
    930 dre-miR-135b 2635 dre-mir-135b
    931 dre-miR-135c 2636 dre-mir-135c-1
    2637 dre-mir-135c-2
    2638 dre-mir-135c-3
    932 fru-miR-135a 2639 fru-mir-135a
    933 fru-miR-135b 2640 fru-mir-135b
    934 tni-miR-135a 2641 tni-mir-135a
    935 tni-miR-135b 2642 tni-mir-135b
    936 mmu-miR-136 2643 mmu-mir-136
    937 rno-miR-136 2644 rno-mir-136
    938 mml-miR-136 2645 mml-mir-136
    939 ggo-miR-136 2646 ggo-mir-136
    940 ppa-miR-136 2647 ppa-mir-136
    941 ptr-miR-136 2648 ptr-mir-136
    942 ppy-miR-136 2649 ppy-mir-136
    943 cfa-miR-136 2650 cfa-mir-136
    944 oar-miR-136 2651 oar-mir-136
    945 ssc-miR-136 2652 ssc-mir-136
    946 mmu-miR-142-5p 2653 mmu-mir-142
    947 rno-miR-142-5p 2654 rno-mir-142
    948 mml-miR-142-5p 2655 mml-mir-142
    949 xtr-miR-142-5p 2656 xtr-mir-142-1
    2657 xtr-mir-142-2
    950 dre-miR-142b-5p 2658 dre-mir-142b
  • [Table 3-20]
    sequence number miRNA name sequence number miRNA precusor name
    951 fru-miR-142a 2659 fru-mir-142a
    952 fru-miR-142b 2660 fru-mir-142b
    953 tni-miR-142a 2661 tni-mir-142a
    954 tni-miR-142b 2662 tni-mir-142b
    955 mmu-miR-146a 2663 mmu-mir-146a
    956 rno-miR-146a 2664 rno-mir-146a
    957 mml-miR-146a 2665 mml-mir-146a
    958 cfa-miR-146a 2666 cfa-mir-146a
    959 gga-miR-146a 2667 gga-mir-146a
    960 dre-miR-146a 2668 dre-mir-146a
    961 mmu-miR-146b 2669 mmu-mir-146b
    962 rno-miR-146b 2670 rno-mir-146b
    963 cfa-miR-146b 2671 cfa-mir-146b
    964 gga-miR-146b 2672 gga-mir-146b
    965 xtr-miR-146b 2673 xtr-mir-146b
    966 dre-miR-146b 2674 dre-mir-146b-1
    2675 dre-mir-146b-2
    967 mmu-miR-181a 2676 mmu-mir-181a-1
    2677 mmu-mir-181a-2
    968 mmu-miR-181c 2678 mmu-mir-181c
    969 mmu-miR-181d 2679 mmu-mir-181d
    970 rno-miR-181a 2680 rno-mir-181a-1
    2681 rno-mir-181a-2
    971 rno-miR-181b 2682 rno-mir-181b-1
    2683 rno-mir-181b-2
    972 rno-miR-181c 2684 rno-mir-181c
    973 rno-miR-181d 2685 rno-mir-181d
    974 lla-miR-181 a 2686 lla-mir-181a-1
    975 lla-miR-181b 2687 lla-mir-181b
    976 sla-miR-181a 2688 sla-mir-181a-2
    977 mml-miR-181a 2689 mml-mir-181a-1
    2690 mml-mir-181a-2
    978 mml-miR-181b 2691 mml-mir-181b-1
    2692 mml-mir-181b-2
    979 mml-miR-181c 2693 mml-mir-181c
    980 mml-miR-181d 2694 mml-mir-181d
    981 mne-miR-181a 2695 mne-mir-181a-1
    2696 mne-mir-181a-2
    982 mne-miR-181b 2697 mne-mir-181b
    983 ggo-miR-181a 2698 ggo-mir-181a-1
    2699 ggo-mir-181a-2
    984 ggo-miR-181b 2700 ggo-mir-181b
    985 ggo-miR-181c 2701 ggo-mir-181c
    986 ppa-miR-181a 2702 ppa-mir-181a-1
    2703 ppa-mir-181a-2
    987 ppa-miR-181b 2704 ppa-mir-181b
    988 ppa-miR-181c 2705 ppa-mir-181c
    989 ptr-miR-181a 2706 ptr-mir-181a-1
    2707 ptr-mir-181a-2
    990 ptr-miR-181b 2708 ptr-mir-181b
    991 ptr-miR-181c 2709 ptr-mir-181c
    992 ppy-miR-181a 2710 ppy-mir-181a-1
    993 ppy-miR-181b 2711 ppy-mir-181b
    994 cfa-miR-181a 2712 cfa-mir-181a-1
    2713 cfa-mir-181a-2
    995 cfa-miR-181b 2714 cfa-mir-181b-1
    2715 cfa-mir-181b-2
    996 cfa-miR-181c 2716 cfa-mir-181c
    997 cfa-miR-181d 2717 cfa-mir-181d
    998 mdo-miR-181a 2718 mdo-mir-181a
    999 mdo-miR-181b 2719 mdo-mir-181b
    1000 mdo-miR-181c 2720 mdo-mir-181c
  • [Table 3-21]
    sequence number miRNA name sequence number miRNA precusor name
    1001 gga-miR-181a 2721 gga-mir-181a-1
    2722 gga-mir-181a-2
    1002 gga-miR-181b 2723 gga-mir-181b-1
    2724 gga-mir-181b-2
    1003 xtr-miR-181a 2725 xtr-mir-181a-1
    2726 xtr-mir-181a-2
    1004 xtr-miR-181b 2727 xtr-mir-181b-1
    2728 xtr-mir-181b-2
    1005 bta-miR-181a 2729 bta-mir-181a
    1006 bta-miR-181 b 2730 bta-mir-181b
    1007 bta-miR-181c 2731 bta-mir-181c
    1008 ssc-miR-181b 2732 ssc-mir-181b
    1009 ssc-miR-181c 2733 ssc-mir-181c
    1010 dre-miR-181a 2734 dre-mir-181a-1
    2735 dre-mir-181a-2
    1011 dre-miR-181b 2736 dre-mir-181b-1
    2737 dre-mir-181b-2
    1012 dre-miR-181c 2738 dre-mir-181 c
    1013 fru-miR-181a 2739 fru-mir-181a-1
    2740 fru-mir-181a-2
    1014 fru-miR-181b 2741 fru-mir-181b-1
    2742 fru-mir-181b-2
    1015 tni-miR-181a 2743 tni-mir-181a-1
    2744 tni-mir-181a-2
    1016 tni-miR-181b 2745 tni-mir-181b-1
    2746 tni-mir-181b-2
    1017 xtr-miR-182* 2747 xtr-mir-182
    1018 dre-miR-182* 2748 dre-mir-182
    1019 mmu-miR-183 2749 mmu-mir-183
    1020 rno-miR-183 2750 rno-mir-183
    1021 sla-miR-183 2751 sla-mir-183
    1022 mml-miR-183 2752 mml-mir-183
    1023 mne-miR-183 2753 mne-mir-183
    1024 ggo-miR-183 2754 ggo-mir-183
    1025 ppa-miR-183 2755 ppa-mir-183
    1026 ptr-miR-183 2756 ptr-mir-183
    1027 cfa-miR-183 2757 cfa-mir-183
    1028 mdo-miR-183 2758 mdo-mir-183
    1029 gga-miR-183 2759 gga-mir-183
    1030 xtr-miR-183 2760 xtr-mir-183
    1031 ssc-miR-183 2761 ssc-mir-183
    1032 dre-miR-183 2762 dre-mir-183
    1033 fru-miR-183 2763 fru-mir-183
    1034 tni-miR-183 2764 tni-mir-183
    1035 mmu-miR-187 2765 mmu-mir-187
    1036 rno-miR-187 2766 rno-mir-187
    1037 mml-miR-187 2767 mml-mir-187
    1038 mne-miR-187 2768 mne-mir-187
    1039 ggo-miR-187 2759 ggo-mir-187
    1040 ppa-miR-187 2770 ppa-mir-187
    1041 ppy-miR-187 2771 ppy-mir-187
    1042 mdo-miR-187 2772 mdo-mir-187
    1043 gga-miR-187 2773 gga-mir-187
    1044 xtr-miR-187 2774 xtr-mir-187
    1045 dre-miR-187 2775 dre-mir-187
    1046 fru-miR-187 2776 fru-mir-187
    1047 tni-miR-181 2777 tni-mir-187
    1048 mmu-miR-192 2778 mmu-mir-192
    1049 rno-miR-132 2779 rno-mir-192
    1050 mmi-miR-192 2780 mml-mir-192
  • [Table 3-22]
    sequence number miRNA name sequence number miRNA precusor name
    1051 cfa-miR-192 2781 cfa-mir-192
    1052 xtr-miR-192 2782 xtr-mir-192
    1053 bta-miR-192 2783 bta-mir-192
    1054 dre-miR-192 2784 dre-mir-192
    1055 fru-miR-192 2785 fru-mir-192
    1056 tni-miR-192 2786 tni-mir-192
    1057 mmu-miR-215 2387 mmu-mir-215
    1058 rno-miR-215 2788 rno-mir-215
    1059 mml-miR-215 2789 mml-mir-215
    1060 mne-miR-215 2790 mne-mir-215
    1061 ggo-miR-215 2791 ggo-mir-215
    1062 ptr-miR-215 2792 ptr-mir-215
    1063 ppy-miR-215 2793 ppy-mir-215
    1064 gga-miR-215 2794 gga-mir-215
    1065 xtr-miR-215 2795 xtr-mir-215
    1066 bta-miR-215 2796 bta-mir-215
    1067 mmu-miR-200a* 2797 mmu-mir-200a
    1068 mdo-miR-200a* 2798 mdo-mir-200a
    1069 rno-miR-216a 2799 rno-mir-216a
    1070 mml-miR-216a 2800 mml-mir-216a
    1071 ggo-miR-216 2801 ggo-mir-216
    1072 ppa-miR-216 2802 ppa-mir-216
    1073 ppy-miR-216 2803 ppy-mir-216
    1074 lca-miR-216 2804 lca-mir-216
    1075 mdo-miR-216 2805 mdo-mir-216
    1076 gga-miR-216 2806 gga-mir-216
    1077 xtr-miR-216 2807 xtr-mir-216
    1078 ssc-miR-216 2808 ssc-mir-216
    1079 dre-miR-216a 2809 dre-mir-216a-1
    2810 dre-mir-216a-2
    1080 fru-miR-216a 2811 fru-mir-216a
    1081 tni-miR-216a 2812 tni-mir-216a
    1082 mmu-miR-217 2813 mmu-mir-217
    1083 rno-miR-217 2814 rno-mir-217
    1084 ggo-miR-217 2815 ggo-mir-217
    1085 ppa-miR-217 2816 ppa-mir-217
    1086 mdo-miR-217 2817 mdo-mir-217
    1087 gga-miR-217 2818 gga-mir-217
    1088 xtr-miR-217 2819 xtr-mir-217
    1089 ssc-miR-217 2820 ssc-mir-217
    1090 dre-miR-217 2821 dre-mir-217-1
    2822 dre-mir-217-2
    1091 fru-miR-217 2823 fru-mir-217
    1092 tni-miR-217 2824 tni-mir-217
    1093 ggo-miR-220 2825 ggo-mir-220
    1094 ppa-miR-220 2826 ppa-mir-220
    1095 ptr-miR-220 2827 ptr-mir-220
    1096 mmu-miR-222 2828 mmu-mir-222
    1097 rno-miR-222 2829 rno-mir-222
    1098 age-miR-222 2830 age-mir-222
    1099 mml-miR-222 2831 mml-mir-222
    1100 gga-miR-222 2832 gga-mir-222
  • [Table 3-23]
    sequence number miRNA name sequence number miRNA precursor name
    1101 xtr-miR-222 2833 xtr-mir-222
    1102 bta-miR-222 2834 bta-mir-222
    1103 dre-miR-222 2835 dre-mir-222
    1104 fru-miR-222 2836 fru-mir-222
    1105 tni-miR-222 2837 tni-mir-222
    1106 mmu-miR-223 2838 mmu-mir-223
    1107 rno-miR-223 2839 rno-mir-223
    1108 sla-miR-223 2840 sla-mir-223
    1109 mml-miR-223 2841 mml-mir-223
    1110 ggo-miR-223 2842 ggo-mir-223
    1111 ppa-miR-223 2843 ppa-mir-223
    1112 ptr-miR-223 2844 ptr-mir-223
    1113 ppy-miR-223 2845 ppy-mir-223
    1114 mdo-miR-223 2846 mdo-mir-223
    1115 gga-miR-223 2847 gga-mir-223
    1116 xtr-miR-223 2848 xtr-mir-223
    1117 dre-miR-223 2849 dre-mir-223
    1118 fru-miR-223 2850 fru-mir-223
    1119 tni-miR-223 2851 tni-mir-223
    1120 mmu-miR-224 2852 mmu-mir-224
    1121 rno-miR-224 2853 rno-mir-224
    1122 mml-miR-224 2854 mml-mir-224
    1123 mne-miR-224 2855 mne-mir-224
    1124 ggo-miR-224 2856 ggo-mir-224
    1125 ppa-miR-224 2857 ppa-mir-224
    1126 ptr-miR-224 2858 ptr-mir-224
    1127 ppy-miR-224 2859 ppy-mir-224
    1128 cfa-miR-224 2860 cfa-mir-224
    1129 ssc-miR-224 2861 ssc-mir-224
    1130 mmu-miR-302b* 2862 mmu-mir-302b
    1131 gga-miR-302b* 2863 gga-mir-302b
    1132 gga-miR-302c* 2864 gga-mir-302c
    1133 mmu-miR-374 2865 mmu-mir-374
    1134 rno-miR-374 2866 rno-mir-374
    1135 mml-miR-374a 2867 mml-mir-374a
    1136 mml-miR-374b 2868 mml-mir-374b
    1137 cfa-miR-374b 2869 cfa-mir-374b
    1138 bta-miR-374 2870 bta-mir-374
    1139 rno-miR-376b-3p 2871 rno-mir-376b
    1140 mml-miR-376a 2872 mml-mir-376a-1
    2873 mml-mir-376a-2
    1141 mml-miR-376b 2874 mml-mir-376b
    1142 cfa-miR-376 2875 cfa-mir-376-1
    2876 cfa-mir-376-2
    2877 cfa-mir-376-3
    1143 mmu-miR-409-5p 2878 mmu-mir-409
    1144 rno-miR-409-5p 2879 rno-mir-409
    1145 mml-miR-409-5p 2880 mml-mir-409
    1146 mmu-miR-429 2881 mmu-mir-429
    1147 rno-miR-429 2882 rno-mir-429
    1148 mml-miR-429 2883 mml-mir-429
    1149 cfa-miR-429 2884 cfa-mir-429
    1150 gga-miR-429 2885 gga-mir-429
  • [Table 3-24]
    sequence number miRNA name sequence number miRNA precursor name
    1151 xla-miR-429 2886 xla-mir-429
    1152 xtr-miR-429 2887 xtr-mir-429
    1153 dre-miR-429 2888 dre-mir-429
    1154 fru-miR-429 2889 fru-mir-429
    1155 tni-miR-429 2890 tni-mir-429
    1156 mmu-miR-200c 2891 mmu-mir-200c
    1157 mo-miR-200b 2892 mo-mir-200b
    1158 mo-miR-200c 2893 mo-mir-200c
    1159 cfa-miR-200c 2894 cfa-mir-200c
    1160 mdo-miR-200b 2895 mdo-mir-200b
    1161 mdo-miR-200c 2896 mdo-mir-200c
    1162 gga-miR-200b 2897 gga-mir-200b
    1163 xtr-miR-200b 2898 xtr-mir-200b
    1164 bta-miR-200b 2899 bta-mir-200b
    1165 bta-miR-200c 2900 bta-mir-200c
    1166 dre-miR-200b 2901 dre-mir-200b
    1167 dre-miR-200c 2902 dre-mir-200c
    1168 fru-miR-200b 2903 fru-mir-200b
    1169 tni-miR-200b 2904 tni-mir-200b
    1170 mmu-miR-448 2905 mmu-mir-448
    1171 mo-miR-448 2906 mo-mir-448
    1172 mml-miR-448 2907 mml-mir-448
    1173 cfa-miR-448 2908 cfa-mir-448
    1174 cfa-miR-450a 2909 cfa-mir-450a
    1175 mmu-miR-451 2910 mmu-mir-451
    1176 mo-miR-451 2911 mo-mir-451
    1177 mml-miR-451 2912 mml-mir-451
    1178 mdo-miR-451 2913 mdo-mir-451
    1179 gga-miR-451 2914 gga-mir-451
    1180 xtr-miR-451 2915 xtr-mir-451
    1181 dre-miR-451 2916 dre-mir-451
    1182 mmu-miR-452 2917 mmu-mir-452
    1183 mmu-miR-487b 2918 mmu-mir-487b
    1184 mo-miR-487b 2919 mo-mir-487b
    1185 mml-miR-487b 2920 mml-mir-487b
    1186 bta-miR-487b 2921 bta-mir-487b
    1187 gga-miR-489 2922 gga-mir-489
    1188 xtr-miR-489 2923 xtr-mir-489
    1189 dre-miR-489 2924 dre-mir-489
    1190 fru-miR-489 2925 fru-mir-489
    1191 tni-miR-489 2926 tni-mir-489
    1192 age-miR-514 2927 age-mir-514
    1193 mml-miR-514 2928 mml-mir-514-1
    2929 mml-mir-514-2
    1194 pbi-miR-514 2930 pbi-mir-514
    1195 tr-miR-514 2931 ptr-mir--514-1
    2932 ptr-mir-514-2
    2933 ptr-mir-514-3
    2934 ptr-mir-514-4
    1196 ssy-miR-514 2935 ssy-mir-514
    1197 mml-miR-518c 2936 mml-mir-518c
    1198 mml-miR-518d 2937 mml-mir-518d
    1199 mml-miR-542-3p 2938 mmu-mir-542
    1200 mo-miR-542-3p 2939 mo-mir-542
  • [Table 3-25]
    sequence number miRNA name sequence number miRNA precursor name
    1201 mml-miR-542-3p 2940 mml-mir-542
    1202 mmu-miR-544 2941 mmu-mir-544
    1203 mml-miR-544 2942 mml-mir-544
    1204 mml-miR-601 2943 mml-mir-601
    1205 mml-miR-609 2944 mml-mir-609
    1206 mml-miR-632 2945 mml-mir-532
    1207 mml-miR-644 2946 mml-mir-644
    1208 mmu-miR-365 2947 mmu-mir-365-1
    2948 mmu-mir-365-2
    1209 mo-miR-365 2949 mo-mir-365
    1210 mml-miR-365 2950 mml-mir-365-1
    2951 mml-mir-365-2
    1211 cfa-miR-365 2952 cfa-mir-365-1
    2953 cfa-mir-365-2
    1212 mdo-miR-365 2954 mdo-mir-365
    1213 gga-miR-365 2955 gga-mir-365-1
    2956 gga-mir-365-2
    1214 xtr-miR-365 2957 xtr-mir-365-1
    1215 bta-miR-365 2958 bta-mir-365
    1216 dre-miR-365 2959 dre-mir-365-1
    2960 dre-mir-365-2
    2961 dre-mir-365-3
    2962 dre-mir-365-4
    1217 fru-miR-365 2963 fru-mir-365
    1218 tni-miR-365 2964 tni-mir-365
    1219 mmu-miR-142-3p 2653 mmu-mir-142
    1220 mo-miR-142-3p 2654 mo-mir-142
    1221 mml-miR-142-3p 2655 mml-mir-142
    1222 gga-miR-142-3p 2965 gga-mir-142
    1223 xtr-miR-142-3p 2656 xtr-mir-142-1
    2657 xtr-mir-142-2
    1224 mmu-miR-200a 2797 mmu-mir-200a
    1225 mo-miR-200a 2966 mo-mir-200a
    1226 mml-miR-200a 2967 mml-mir-200a
    1227 mdo-miR-200a 2798 mdo-mir-200a
    1228 gga-miR-200a 2968 gga-mir-200a
    1229 xtr-miR-200a 2969 xtr-mir-200a
    1230 bta-miR-200a 2970 bta-mir-200a
    1231 dra-miR-200a 2971 dre-mir-200a
    1232 fru-miR-200a 2972 fru-mir-200a
    1283 tni-miR-200a 2973 tni-mir-200a
    1234 mmu-miR-361 2974 mmu-mir-361
    1235 mo-miR-361 2975 mo-mir-361
    1236 cfa-miR-361 2976 cfa-mir-361
    1237 bta-miR-361 2977 bta-mir-361
    1238 hsa-miR-15a 2978 hsa-mir-15a
    1239 hsa-miR-15b 2979 hsa-mir-15b
    1240 hsa-miR-424 2980 hsa-mir-424
    1241 hsa-miR-497 2981 hsa-mir-497
    1242 mmu-miR-15a 2982 mmu-mir-15a
    1243 mmu-miR-15b 2983 mmu-mir-15b
    1244 mmu-miR-322 2984 mmu-mir-322
    1245 mmu-miR-497 2985 mmu-mir-497
    1246 mo-miR-15b 2986 mo-mir-15b
    1247 mo-miR-322 2987 mo-mir-322
    1248 mo-miR-497 2988 mo-mir-497
    1249 age-miR-15a 2989 age-mir-15a
    1250 age-miR-15b 2990 age-mir-15b
  • [Table 3-26]
    sequence number miRNA name sequence number miRNA precursor name
    1251 IIa-miR-15a 2991 IIa-mir-15a
    1252 IIa-miR-15b 2992 IIa-mir-15b
    1253 sla-miR-15a 2993 sla-mir-15a
    1254 mml-miR-15a 2994 mml-mir-15a
    1255 mml--miR-15b 2995 mml-mir-15b
    1256 mml-miR-424 2996 mml-mir-424
    1257 mml-miR-497 2997 mml-mir-497
    1258 mne-miR-15a 2998 mne-mir-15a
    1259 mne-miR-15b 2999 mne-mir-15b
    1260 ggo-miR-15a 3000 ggo-mir-15a
    1261 ggo-miR-15b 3001 ggo-mir-15b
    1262 ppa-miR-15a 3002 ppa-mir-15a
    1263 ppa-miR-15b 3003 ppa-mir-15b
    1264 ptr-miR-15a 3004 ptr-mir-15a
    1265 ptr-miR-15b 3005 ptr-mir-15b
    1266 ppy-miR-15a 3006 ppy-mir-15a
    1267 ppy-miR-15b 3007 ppy-mir-15b
    1268 lca-miR-15a 3008 lca-mir-15a
    1269 cfa-miR-15a 3009 cfa-mir-15a
    1270 cfa-miR-15b 3010 cfa-mir-15b
    1271 cfa-miR-497 3011 cfa-mir-497
    1272 mdo-miR-15a 3012 mdo-mir-15a
    1273 gga-miR-15a 3013 gga-mir-15a
    1274 gga-miR-15b 3014 gga-mir-15b
    1275 xtr-miR-15a 3015 xtr-mir-15a
    1276 xtr-miR-15b 3016 xtr-mir-15b
    1277 xtr-miR-15c 3017 xtr-mir-15c
    1278 bta-miR-15a 3018 bta-mir-15a
    1279 bta-miR-15b 3019 bta-mir-15b
    1280 bta-miR-497 3020 bta-mir-497
    1281 dre-miR-15a 3021 dre-mir-15a-1
    3022 dre-mir-15a-2
    1282 dre-miR-15b 3023 dre-mir-15b
    1283 dre-miR-457a 3024 dre-mir-457a
    1284 dre-miR-457b 3025 dre-mir-457b
    1285 fru-miR-15a 3026 fru-mir-15a
    1285 tni-miR-15a 3027 tni-mir-15a
    1287 mmu-miR-20b* 1786 mmu-mir-20b
    1288 mo-miR-20b-3p 3028 mo-mir-20b
    1289 dre-miR-20a* 1809 dre-mir-20a
    1290 hsa-miR-590-5p 3029 hsa-mir-590
    1291 mmu-miR-590-5p 3030 mmu-mir-590
    1292 mml-miR-590-5p 3031 mml-mir-590
    1293 hsa-miR-92a 3032 hsa-mir-92a-1
    3033 hsa-mir-92a-2
    1294 hsa-miR-92b 3034 hsa-mir-92b
    1295 hsa-miR-363 3035 hsa-mir-363
    1296 hsa-miR-367 3036 hsa-mir-367
    1297 mmu-miR-92a 3037 mml-mir-92a-1
    3038 mmu-mir-92a-2
    1298 mnu-miR-92b 3039 mmu-mir-92b
    1299 mmu-miR-363 3040 mmu-mir-363
    1300 mmu-miR-367 3041 mmu-mir-367
  • [Table 3-27]
    sequence number miRNA name sequence number miRNA precursor name
    1301 mo-miR-92a 3042 mo-mir-92a-1
    3043 mo-mir-92a-2
    1302 mo-miR-92b 3044 mo-mir-92b
    1303 mo-miR-363 3045 mo-mir-363
    1304 age-miR-92 3046 age-mir-92-1
    3047 age-mir-92-2
    1305 IIa-miR-92 3048 IIa-mir-92-1
    3049 IIa-mir-92-2
    1306 sla-miR-92 3050 sla-mir-92-1
    3051 sla-mir-92-2
    1307 mml-miR-92a 3052 mml-mir-92a-1
    3053 mml-mir-92a-2
    1308 mml-miR-92b 3054 mml-mir-92b
    1309 mml-miR-363 3055 mml-mir-363
    1310 mml-miR-367 3056 mml-mir-367
    1311 mne-miR-92 3057 mne-mir-92
    1312 ggo-miR-32 3058 ggo-mir-92-1
    3059 ggo-mir-92-2
    1313 ppa-mir-92 3060 ppa-mir-92-1
    3061 ppa-mir-92-2
    1314 ptr-miR-92 3062 ptr-mir-92-1
    3063 ptr-mir-92-2
    1315 ppy-mir-92 3064 ppy-mir-92-1
    3065 ppy-mir-92-2
    1316 lca-miR-92 3066 lca-mir-92-1
    3067 lca-mir-92-2
    1317 cfa-miR-92a 3068 cfa-mir-92a-1
    3069 cfa-mir-92a-2
    1318 cfa-miR-92b 3070 cfa-mir-92b
    1319 cfa-miR-363 3071 cfa-mir-363
    1320 mdo-miR-92 3072 mdo-mir-92
    1321 gga-miR-92 3073 gga-mir-92
    1322 gga-miR-367 3074 gga-mir-367
    1323 xtr-miR-92a 3075 xtr-mir-92a-1
    3076 xtr-mir-92a-2
    1324 xtr-miR-92b 3077 xtr-mir-92b
    1325 xtr-miR-363-3p 3078 xtr-mir-363
    1326 xtr-miR-367 3079 xtr-mir-367
    1327 bta-miR-32 3080 bta-mir-92
    1328 dre-miR-92a 3081 dre-mir-92a-1
    3082 dre-mir-92a-2
    1329 dre-miR-92b 3083 dre-mir-92b
    1330 dre-miR-363 3084 dre-mir-363
    1331 fru-miR-92 3085 fru-mir-92-1
    3086 fru-mir-92-2
    1332 tni-miR-92 3087 tni-mir-92-1
    3088 tni-mir-92-2
    1333 hsa-miR-26a 3089 hsa-mir-26a-1
    3090 hsa-mir-26a-2
    1334 hsa-miR-1297 3091 hsa-mir-1297
    1335 hsa-miR-30d* 3092 hsa-mir-30d
    1336 hsa-miR-30e* 3093 hsa-mir-30e
    1337 hsa-miR-34c-5p 3094 hsa-mir-34c
    1338 mmu-miR-699 3095 mmu-mir-699
    1339 hsa-miR-103 3096 hsa-mir-103-1
    3097 hsa-mir-103-2
    1340 mmu-miR-103 3098 mmu-mir-103-1
    3099 mmu-mir-103-2
    1341 mo-miR-103 3100 mo-mir-103-1
    3101 mo-mir-103-2
    1342 age-miR-103 3102 age-mir-103
    1343 lla-miR-103 3103 lla-mir-103
    1344 mml-miR-103 3104 mml-mir-103-1
    3105 mml-mir-103-2
    1345 mne-miR-103 3106 mne-mir-103
    1346 ggo-miR-103 3107 ggo-mir-103
    1347 ppa-miR-103 3108 ppa-mir-103
    1348 ptr-miR-103 3109 ptr-mir-103
    1349 ppy-miR-103 3110 ppy-mir-103
    1350 cfa-miR-103 3111 cfa-mir-103
  • [Table 3-28]
    sequence number miRNA name sequence number miRNA precursor name
    1351 mdo-miR-103 3112 mdo-mir-103-1
    3113 mdo-mir-103-2
    1352 gga-miR-103 3114 gga-mir-103-1
    3115 gga-mir-103-2
    1353 xtr-miR-103 3116 xtr-mir-103-1
    3117 xtr-mir-103-2
    1354 bta-miR-103 3118 bta-mir-103-1
    3119 bta-mir-103-2
    1355 ssc-miR-103 3120 ssc-mir-103
    1356 dre-miR-103 3121 dre-mir-103
    1357 fru-miR-103 3122 fru-mir-103
    1358 tni-miR-103 3123 tni-mir-103
    1359 hsa-miR-148a 3124 hsa-mir-148a
    1360 hsa-miR-152 3125 hsa-mir-152
    1361 mmu-miR-152 3126 mmu-mir-152
    1362 mo-miR-152 3127 mo-mir-152
    1363 mml-miR-152 3128 mml-mir-152
    1364 cfa-miR-152 3129 cfa-mir-152
    1365 mdo-miR-152 3130 mdo-mir-152
    1366 dre-miR-152 3131 dre-mir-152
    1367 fru-miR-152 3132 fru-mir-152
    1368 tni-miR-152 3133 tni-mir-1 52
    1369 hsa-miR-190b 3134 hsa-mir-190b
    1370 hsa-miR-208b 3135 hsa-mir-208b
    1371 dre-miR-736 3136 dre-mir-736
    1372 hsa-miR-204 3137 hsa-mir-204
    1373 mmu-miR-204 3138 mmu-mir-204
    1374 mo-miR-204 3139 mo-mir-204
    1375 sla-miR-204 3140 sla-mir-204
    1376 mml-miR-204 3141 mml-mir-204
    1377 mne-miR-204 3142 mne-mir-204
    1378 ggo-miR-204 3143 ggo-mir-204
    1379 ppa-miR-204 3144 ppa-mir-204
    1380 ptr-miR-204 3145 ptr-mir-204
    1381 ppy-miR-204 3146 ppy-mir-204
    1382 cfa-miR-204 3147 cfa-mir-204
    1383 mdo-miR-204 3148 mdo-mir-204
    1384 gga-miR-204 3149 gga-mir-204-1
    3150 gga-mir-204-2
    1385 xtr-miR-204 3151 xtr-mir-204-1
    3152 xtr-mir-204-2
    1386 bta-miR-204 3153 bta-mir-204
    1387 ssc-miR-204 3154 ssc-mir-204
    1388 dre-miR-204 3155 dre-mir-204-1
    3156 dre-mir-204-2
    3157 dre-mir-204-3
    1389 fru-miR-204 3158 fru-mir-204
    1390 tni-miR-204a 3159 tni-mir-204a
    1391 tni-miR-204b 3160 tni-mir-204b
    1392 mmu-miR-761 3161 mmu-mir-761
    1393 hsa-miR-362-3p 1667 hsa-mir-362
    1394 mmu-miR-362-3p 3162 mmu-mir-362
    1395 mml-miR-362-3p 3163 mml-mir-362
    1396 mmu-miR-489 3164 mmu-mir-489
    1397 dre-miR-731 3165 dre-mir-731
    1398 mo-miR-344-5p 3166 mo-mir-344-1
    1399 hsa-miR-33b* 1634 hsa-mir-33b
    1400 hsa-miR-519e 3167 hsa-mir-519e
  • [Table 3-29]
    sequence number miRNA name sequence number miRNA precursor name
    1401 hsa-miR-519e* 3167 hsa-mir-519e
    1402 hsa-miR-302a 3168 hsa-mir-302a
    1403 hsa-miR-302b 1665 hsa-mir-302b
    1404 hsa-miR-302c 1666 hsa-mir-302c
    1405 hsa-miR-302d 3169 hsa-mir-302d
    1406 hsa-miR-302e 3170 hsa-mir-302e
    1407 hsa-miR-372 3171 hsa-mir-372
    1408 hsa-miR-373 1668 hsa-mir-373
    1409 hsa-miR-520a-3p 3172 hsa-mir-520a
    1410 hsa-miR-520b 3173 hsa-mir-520b
    1411 hsa-miR-520c-3p 3174 hsa-mir-520c
    1412 hsa-miR-520e 3175 hsa-mir-520e
    1413 mmu-miR-291a-3p 3176 mmu-mir-291a
    1414 mmu-miR-294 3177 mmu-mir-294
    1415 mmu-miR-295 3178 mmu-mir-295
    1416 mmu-miR-302a 3179 mmu-mir-302a
    1417 mmu-miR-302b 2862 mmu-mir-302b
    1418 mmu-miR-302d 3180 mmu-mir-302d
    1419 mo-miR-291a-3p 3181 mo-mir-291a
    1420 age-miR-93 3182 age-mir-93
    1421 lla-miR-93 3183 lla-mir-93
    1422 sla-miR-93 3184 sla-mir-93
    1423 mml-miR-93 3185 mml-mir-93
    1424 mml-miR-302a 3186 mml-mir-302a
    1425 mml-miR-302b 3187 mml-mir-302b
    1426 mml-miR-302c 3188 mml-mir-302c
    1427 mml-miR-302d 3189 mml-mir-302d
    1428 mml-miR-372 3190 mml-mir-372
    1429 mml-miR-373 3191 mml-mir-373
    1430 mml-miR-519a 3192 mml-mir-519a
    1431 mne-miR-93 3193 mne-mir-93
    1432 ggo-miR-93 3194 ggo-mir-93
    1433 ppa-miR-93 3195 ppa-mir-93
    1434 ptr-miR-93 3196 ptr-mir-93
    1435 ppy-miR-93 3197 ppy-mir-93
    1436 cfa-miR-106a 3198 cfa-mir-106a
    1437 mdo-miR-93 3199 mdo-mir-93
    1438 mdo-miR-302a 3200 mdo-mir-302a
    1439 mdo-miR-302b 3201 mdo-mir-302b
    1440 mdo-miR-302c 3202 mdo-mir302c
    1441 mdo-miR-302d 3203 mdo-mir-302d
    1442 gga-miR-302b 2863 gga-mir-302b
    1443 gga-miR-302c 2864 gga-mir-302c
    1444 gga-miR-302d 3204 gga-mir-302d
    1445 xla-miR-427 3205 xla-mir-427
    1446 xla-miR-428 3206 xla-mir-428
    1447 xtr-miR-93a 3207 xtr-mir-93a
    1448 xtr-miR-302 3208 xtr-mir-302
    1449 xtr-miR-428 3209 xtr-mir-428
    1450 dre-miR-430a 3210 dre-mir-430a-1
    3211 dre-mir-430a-2
    3212 dre-mir-431a-3
    3213 dre-mir-430a-4
    3214 dre-mir-430a-5
    3215 dre-mir-430a-6
    3216 dre-mir-430a-7
    3217 dre-mir-430a-8
    3218 dre-mir-430a-9
    3219 dre-mir-430a-10
    3220 dre-mir-430a-11
    3221 dre-mir-430a-12
    3222 dre-mir-430a-13
    3223 dre-mir-430a-14
    3224 dre-mir-430a-15
    3225 dre-mir-430a-16
    3226 dre-mir-430a-17
    3227 dre-mir-430a-18
    3228 dre-mir-430a-19
    3229 dre-mir-430a-20
    3230 dre-mir-430a-21
    3231 dre-mir-430a-22
    3232 dre-mir-430a-23
  • [Table 3-30]
    sequence number miRNA name sequence number miRNA precursor name
    1451 dre-miR-430b 3233 dre-mir-430b-1
    3234 dre-mir-430b-2
    3235 dre-mir-430b-3
    3236 dre-mir-430b-4
    3237 dra-mir-430b-5
    3238 dre-mir-430b-6
    3239 dre-mir-430b-7
    3240 dre-mir-430b-8
    3241 dre-mir-431-9
    3242 dre-mir-430b-10
    3243 dre-mir-430b-11
    3244 dre-mir-430b-12
    3245 dre-mir-430b-13
    3246 dre-mir-430b-14
    3247 dre-mir-430b-15
    3248 dre-mir-430b-16
    3249 dre-mir-430b-17
    3250 dre-mir-430b-18
    3251 dre-mir-430b-18
    3252 dre-mir-430b-20
    3253 dre-mir-430b-21
    3254 dre-mir-430b-22
    3255 dre-mir-430b-23
    1452 dre-miR-430c 3256 dre-mir-430c-1
    3257 dre-mir-430c-2
    3258 dra-mir-430c-3
    3259 dre-mir-430c-4
    3260 dre-mir-430c-5
    3261 dre-mir-430c-6
    3262 dre-mir-430c-7
    3263 dre-mir-430c-8
    3264 dre-mir-430c-9
    3265 dre-mir-430c-10
    3266 dre-mir-430c-11
    3267 dre-mir-430c-12
    3268 dre-mir-430c-13
    3269 dre-mir-430c-14
    3270 dre-mir-430c-15
    3271 dre-mir-430c-16
    3272 dre-mir-430c-17
    3273 dre-mir-430c-18
    3274 dre-mir-430c-19
    3275 dre-mir-430c-20
    3276 dre-mir-430c-21
    1453 dre-miR-430i 3277 dre-mir-430i-1
    3278 dre-mir-430i-2
    3279 dre-mir-430i-3
    1454 dre-miR-430j 3280 dre-mir-430j
    1455 mmu-miR-302c 3281 mmu-mir-302c
    1456 gga-miR-302a 3282 gga-mir-302a
    1457 hsa-miR-520g 3283 hsa-mir-520g
    1458 mml-miR-518e 3284 mr-mir-518e
    1459 hsa-miR-524-3p 1688 hsa-mir-524
    1460 hsa-miR-1285 3285 hsa-mir-1285-3
    3286 hsa-mir-1285-2
    1461 hsa-miR-541 3287 hsa-mir-541
    1462 hsa-let-7a 3288 hsa-let-7a-1
    3289 hsa-let-7a-2
    3290 hsa-let-7a-3
    1463 hsa-let-7b 3291 hsa-let-7b
    1464 hsa-let-7c 3292 hsa-let-7c
    1465 hsa-let-7d 3293 hsa-let-7d
    1466 hsa-let-7d 3294 hsa-let-7f-1
    3295 hsa-let-7f-2
    1467 hsa-let-7g 3296 hse-let-7g
    1468 hsa-let-7i 3297 hse-let-7i
    1469 hsa-miR-98 3298 hsa-mir-98
    1470 mmu-miR-98 3299 mmu-mir-98
    1471 mo-miR-98 3300 mo-mir-98
  • [Table 3-31]
    sequence number miRNA name sequence number miRNA precursor name
    1472 age-miR-98 3301 age-mir-98
    1473 mml-miR-98 3302 mml-mir-98
    1474 ggo-miR-98 3303 ggo-mir-98
    1475 ppa-miR-98 3304 ppa-mir-98
    1476 ptr-miR-98 3305 ptr-mir-98
    1477 ppy-miR-98 3306 ppy-mir-98
    1478 cfa-miR-98 3307 cfa-mir-98
    1479 xtr-miR-98 3308 xtr-mir-98
    1480 bta-miR-98 3309 bta-mir-98
    1481 hsa-miR-23a 3310 hsa-mir-23a
    1482 hsa-miR-130a* 3311 hsa-mir-130a
    1483 hsa-miR-27a 3312 hsa-mir-27a
    1484 hsa-miR-708 3313 hsa-mir-708
    1485 mmu-miR-708 3314 mmu-mir-708
    1486 mo-miR-708 3315 mo-mir-708
    1481 cfa-miR-708 3316 cfa-mir-708
    1488 hsa-miR-33a 3317 hsa-mir-33a
    1489 hsa-miR-1271 3318 hsa-mir-1271
    1490 age-miR-507 3319 age-mir-507
    1491 cfa-miR-1271 3320 cfa-mir-1271
    1492 hsa-miR-99a 3321 hsa-mir-99a
    1493 hsa-miR-99b 3322 hsa-mir-99b
    1494 mmu-miR-99a 3323 mmu-mir-99a
    1495 mm-miR-99b 3324 mmu--mir-99b
    1496 mo-miR-99a 3325 mo-mir-99a
    1497 mo-miR-99b 3326 mo-mir-99b
    1498 Ila-miR-99a 3327 Ila-mir-99a
    1499 mmi-miR-99a 3328 mml-mir-99a
    1500 mml-miR-99b 3329 mml-mir-99b
    1501 mne-miR-99a 3330 mne-mir-99a
    1502 ggo-miR-99a 3331 ggo-mir-99a
    1503 ppa-miR-99a 3332 ppa-mir-99a
    1504 ptr-miR-99a 3333 ptr-mir-89a
    1505 ppy-miR-99a 3334 ppy-mir-99a
    1506 cfa-miR-99a 3335 cfa-mir-99a-1
    3336 cfa-mir-99a-2
    1507 cfa-miR-99b 3337 cfa-mir-99b
    1508 gga-miR-99a 3338 gga-mir-99a
    1509 xtr-miR-99 3339 xtr-mir-99
    1510 bta-miR-99a 3340 bta-mir-99a
    1511 bta-miR-99b 3341 bta-mir-99b
    1512 ssc-miR-99b 3342 ssc-mir-99b
    1513 dre-miR-99 3343 dre-mir-99-1
    3344 dre-mir-99-2
    1514 hsa-miR-128 3345 hsa-mir-128-1
    3346 hsa-mir-128-2
    1515 dre-miR-722 3347 dre-mir-722
    1516 hsa-miR-135a 3348 hsa-mir-135a-1
    3349 hsa-mir-135a-2
    1517 hsa-miR-181b 3350 hsa-mir-181b-1
    3351 hsa-mir-181b-2
    1518 hsa-miR-181c 3352 hsa-mir-181c
    1519 hsa-miR-181d 3353 hsa-mir-181d
    1520 hsa-miR-200b* 3354 hsa-mir-200b
    1521 hsa-miR-302d* 3169 hsa-mir-302d
    1522 hsa-miR-616* 3355 hsa-mir-616
    1523 mmu-miR-294* 3177 mmu-mir-294
    1524 hsa-miR-374b 3356 hsa-mir-374b
    1525 hsa-miR-376b 3357 hsa-mir-376b
    1526 hsa-miR-200b 3354 hsa-mir-200b
    1527 mml-miR-548a 3358 mml-mir-548a
    1528 hsa-miR-517a 1590 hsa-mir-517a
  • [Table 3-32]
    sequence number miRHA name sequence number miRNA precursor name
    1529 hsa-miR-518a-3p 3359 hsa-mir-518a-I
    3360 hsa-mir-518a-2
    1530 hsa-miR-518b 3361 hsa-mir-518b
    1531 hsa-miR-518d-3p 3362 hsa-mir-518d
    1532 hsa-miR-518f 3363 hsa-mir-518f
    1533 hsa-miR-520d-5p 1593 hsa-mir-520d
    1534 mml-miR-518a-5p 3364 mml-mir-518a
    1535 mml-miR-520d-5p 3365 mml-mir-520d
    1536 hsa-miR-200c* 1675 hsa-mir-200c
    1537 nmu-miR-200c* 2891 mmu-mir-200c
    1538 mml-miR-580 3366 mml-mir-580
    1539 hsa-miR-141 3367 hsa-mir-141
    1540 mmu-miR-141 3368 mmu-mir-141
    1541 mo-miR-141 3369 mo-mir-141
    1542 mdo-miR-141 3370 mdo-mir-141
    1543 dre-miR-141 3371 dre-mir-141
    3372 hsa-miR-29a 3742 hsa-mir-29a
    3373 hsa-miR-29b 3743 hsa-mir-29b-1
    3744 hsa-mir-29b-2
    3374 hsa-miR-30a-5p 1557 hsa-mir-30a
    3375 hsa-miR-30b 3745 hsa-mir-30b
    3376 hsa-miR-105 3746 hsa-mir-105-1
    3747 hsa-mir-105-2
    3377 hsa-miR-124a 3748 hsa-mir-124-1
    3749 hsa-mir-124-2
    3750 hsa-mir-124-3
    3378 hsa-miR-128a 3345 hsa-mir-128-1
    3379 hsa-miR-150 3751 hsa-mir-150
    3380 hsa-miR-154 3752 hsa-mir-154
    3381 hsa-miR-299-3p 1575 hsa-mir-299
    3382 hsa-miR-380-5p 3753 hsa-mir-380
    3383 hsa-miR-383 3754 hsa-mir-383
    3384 hsa-miR-411 3755 hsa-mir-411
    3385 hsa-miR-423 3756 hsa-mir-423
    3386 hsa-miR-433 3757 hsa-mir-433
    3387 hsa-miR-454-3p 3758 hsa-mir-454
    3388 hsa-miR-501 3759 hsa-mir-501
    3389 hsa-miR-504 3760 hsa-mir-504
    3390 hsa-miR-506 3761 hsa-mir-506
    3391 hsa-miR-507 3762 hsa-mir-507
    3392 hsa-miR-508 3763 hsa-mir-508
    3393 hsa-miR-511 3764 hsa-mir-511-1
    3765 hsa-mir-511-2
    3394 hsa-miR-512-3p 3766 hsa-mir-512-1
    3767 hsa-mir-512-2
    3395 hsa-miR-527 3768 hsa-mir-527
    3396 hsa-miR-562 3769 hsa-mir-562
    3397 hsa-miR-567 3770 hsa-mir-567
    3398 hsa-miR-589 3771 hsa-mir-589
    3399 hsa-miR-597 3772 hsa-mir-597
    3400 hsa-miR-605 3773 hsa-mir-605
    3401 hsa-miR-619 3774 hsa-mir-619
    3402 hsa-miR-629 3775 hsa-mir-629
    3403 hsa-miR-640 3776 hsa-mir-640
  • [Table 3-33]
    sequence number miRNA name sequence number miRNA precursor name
    3404 hsa-miR-767-5p 3777 hsa-mir-767
    3405 hsa-miR-370-5p 3778 hsa-mir-770
    3406 hsa-miR-802 3779 hsa-mir-802
    3407 xtr-miR-16c 3780 xtr-mir-16c
    3408 mmu-miR-17* 3781 mmu-mir-17
    3409 cfa-miR-17 3782 cfa-mir-17
    3410 dre-miR-17a* 3783 dre-mir-17a-1
    3784 dre-mir-17a-2
    3411 xla-miR-18 3785 xla-mir-18
    3412 mmu-miR-20a 3786 mmu-mir-20a
    3413 mo-miR-20b-5p 3028 mo-mir-20b
    3414 xtr-miR-24a 3787 xtr-mir-24a
    3415 mmu-miR-26a 3788 mmu-mir-26a-1
    3789 mmu-mir-26a-2
    3416 mmu-miR-30a* 3790 mmu-mir-30a
    3417 mmu-miR-30e* 3791 mmu-mir-30e
    3418 mo-miR-30a* 3792 mo-mir-30a
    3419 mmu-miR-34c 3793 mmu-mir-34c
    3420 mmu-miR-148a 3794 mmu-mir-148a
    3421 mo-miR-148b-3p 3795 mo-mir-148b
    3422 mmu-miR-190b 3796 mmu-mir-190b
    3423 mmu-miR-199a-5p 3797 mmu-mir-199a-1
    3798 mmu-mir-199a-2
    3424 mmu-miR-199b* 3799 mmu-mir-199b
    3425 mo-miR-199a-5p 3800 mo-mir-199a
    3426 mml-miR-199a-5p 2016 mml-mir-199a-1
    2017 mml-mir-199a-2
    3427 gga-miR-199 3801 gga-mir-199-1
    3802 gga-mir-199-2
    3428 bta-miR-199a-5p 3803 bta-mir-199a
    3429 mmu-miR-208a 3804 mmu-mir-208a
    3430 dre-miR-218a 3805 dre-mir-218a-1
    3806 dre-mir-218a-2
    3431 mmu-miR-299* 3807 mmu-mir-299
    3432 mo-miR-299 3808 mo-mir-299
    3433 mmu-miR-325* 3809 mmu-mir-325
    3434 mo-miR-325-5p 3810 mo-mir-325
    3435 mmu-miR-338-3p 3811 mmu-mir-338
    3436 mml-miR-338-3p 3812 mml-mir-338
    3437 mmu-miR-425 3813 mmu-mir-425
    3438 mo-miR-425 3814 mo-mir-425
    3439 mml-miR-425 3815 mml-mir-425
    3440 cfa-miR-425 3816 cfa-mir-425
    3441 mmu-miR-485 3817 mmu-mir-485
    3442 mo-miR-4B5 3818 mo-mir-485
    3443 cfa-miR-485 3819 cfa-mir-485
    3444 mml-miR-486-5p 3820 mml-mir-486
    3445 mmu-miR-488* 3821 mmu-mir-488
    3446 mml-miR-520a 3822 mml-mir-520a
    3447 mml-miR-520d-3p 3365 mml-mir-520d
    3448 mml-miR-520g 3823 mml-mir-520g
    3449 mml-miR-650a 3824 mml-mir-650a-1
    3825 mml-mir-650a-2
    3450 mml-miR-654-5p 3826 mml-mir-654
    3451 mmu-miR-675-5p 3827 mmu-mir-675
    3452 dre-miR-194a 3828 dre-mir-194a
    3453 mmu-let-7g 3829 mmu-let-7g
    3454 xtr-miR-1a 3830 xtr-mir-1a-1
    3831 xtr-mir-1a-2
  • [Table 3-34]
    sequence number miRNA name sequence number miRNA precursor name
    3455 xtr-miR-9a 3832 xtr-mir-9a-1
    3833 xtr-mir-9a-2
    3456 ssc-miR-9-1 3834 ssc-mir-9-1
    3835 ssc-mir-9-2
    3457 ssc-miR-9-2 3834 ssc-mir-9-1
    3835 ssc-mir-9-2
    3458 mmu-miR-23a 3836 mmu-mir-23a
    3459 mmu-miR-27a 3837 mmu-mir-27a
    3460 xtr-miR-31b 3838 xtr-mir-31b
    3461 mmu-miR-33 3839 mmu-mir-33
    3462 mmu-miR-106b 3840 mmu-mir-106b
    3463 mmu-miR-126-5p 3841 mmu-mir-126
    3464 cfa-miR-126 3842 cfa-mir-126
    3465 mmu-miR-128 3843 mmu-mir-128-1
    3844 mmu-mir-128-2
    3466 mmu-miR-129-5p 3845 mmu-mir-129-1
    3846 mmu-mir-129-2
    3467 mml-miR-129-5p 3847 mml-mir-129
    3468 xtr-miR-133c 3848 xtr-mir-133c
    3469 mmu-miR-135a 3849 mmu-mir-135a-1
    3850 mmu-mir-135a-2
    3470 bta-miR-142 3851 bta-mir-142
    3471 dre-miR-142a-5p 3852 dre-mir-142a
    3472 mml--miR-146b-5p 3853 mml-mir-146b
    3473 xtr-miR-146 3854 xtr-mir-146
    3474 mmu-miR-181b 3855 mmu-mir-181b-1
    3856 mmu-mir-181 b-2
    3475 fru-miR-182 3857 fru-mir-182
    3476 tni-miR-182 3858 tni-mir-182
    3477 mmu-miR-200b* 3859 mmu-mir-200b
    3478 mmu-miR-216a 3860 mmu-mir-216a
    3479 mdo-miR-222a 3861 mdo-mir-222a
    3480 mmu-miR-296-5p 3862 mmu-mir-296
    3481 mo-miR-296* 3863 mo-mir-296
    3482 rnml-miR-296-5p 3864 mml-mir-296
    3483 mmu-miR-302c* 3281 mmu-mir-302c
    3484 mmu-miR-362-5p 3162 mmu-mir-362
    3485 mml-miR-362-5p 3163 mml-mir-362
    3486 cfa-miR-374a 3865 cfa-mir-374a
    3487 mmu-mR-376b 3866 mmu-mir-376b
    3488 mmu-miR-378* 3867 mmu-mir-378
    3489 mo-miR-378* 3868 mo-mir-378
    3490 mmu-miR-200b 3859 mmu-mir-200b
    3491 mo-miR-450a 3869 mo-mir-450a
    3492 cfa-miR-487 3870 cfa-mir-487
    3493 mml-miR-517a 3871 mml-mir-517a
    3494 mml-miR-518b 3872 mml-mir-518b
    3495 cfa-miR-542 3873 cfa-mir-542
    3496 mdo-miR-142 3874 mdo-mir-142
    3497 cfa-miR-142a-3p 3852 dre-mir-142a
    3498 mml-miR-361-5p 3875 mml-mir-361
    3499 mmu-miR-29a 3876 mmu-mir-29a
    3500 mmu-miR-29b 3877 mmu-mir-29b-1
    3878 mmu-mir-29b-2
    3501 mmu-miR-29c 3879 mmu-mir-29c
    3502 mo-miR-29a 3880 mo-mir-29a
  • [Table 3-35]
    sequence number miRHA name sequence number miRNA precursor name
    3503 mo-miR-29b 3881 mo-mir-29b-1
    3882 mo-mir-29b-2
    3504 mo-miR-29c 3883 mo-mir-29c
    3505 age-miR-29b 3884 age-mir-29b
    3506 Ila-miR-29b 3885 Ila-mir-29b
    3507 sla-miR-29b 3886 sla-mir-29b
    3508 mml-miR-29b 3887 mml-mir-29b-1
    3888 mml-mir-29b-2
    3509 mml-miR-29c 3889 mml-mir-29c
    3510 mne-miR-29b 3890 mne-mir-29b
    3511 ggo-miR-29b 3891 ggo-mir-29b-1
    3892 ggo-mir-29b-2
    3512 ppa-miR-29b 3893 ppa-mir-29b-1
    3894 ppa-mir-29b-2
    3513 ptr-miR-29b 3895 ptr-mir-29b-1
    3896 ptr-mir-29b-2
    3514 ppy-miR-29b 3897 ppy-mir-29b-1
    3898 ppy-mir-29b-2
    3515 cfa-miR-29a 3899 cfa-mir-29a
    3516 cfa-miR-29b 3900 cfa-mir-29b-1
    3901 cfa-mir-29b-2
    3517 cfa-miR-29c 3902 cfa-mir-29c
    3518 mdo-miR-29a 3903 mdo-mir-29a
    3519 mdo-miR-29b 3904 mdo-mir-29b
    3520 gga-miR-29a 3905 gga-mir-29a
    3521 gga-miR-29b 3906 gga-mir-29b-1
    3907 gga-mir-29b-2
    3522 gga-miR-29c 3908 gga-mir-29c
    3523 xtr-miR-29a 3909 xtr-mir-29a
    3524 xtr-miR-29b 3910 xtr-mir-29b
    3525 xtr-miR-29c 3911 xtr-mir-29c
    3526 xtr-miR-29d 3912 xtr-mir-29d
    3527 bta-miR-29b 3913 bta-mir-29b
    3528 bta-miR-29c 3914 bta-mir-29c
    3529 ssc-miR-29b 3915 ssc-mir-29b
    3530 ssc-miR-29c 3916 ssc-mir-29c
    3531 dre-miR-29a 3917 dre-mir-29a-1
    3918 dre-mir-29a-2
    3532 dre-miR-29b 3919 dre-mir-29b-1
    3920 dre-mir-29b-2
    3921 dre-mir-29b-3
    3533 fru-miR-29a 3922 fru-mir-29a-1
    3923 fru-mir-29a-2
    3534 fru-miR-29b 3924 fru-mir-29b-1
    3925 fru-mir-29b-2
    3535 tni-miR-29a 3926 tni-mir-29a-1
    3927 tni-mir-29a-2
    3536 tni-miR-29b 3928 tni-mir-29b-1
    3929 tni-mir-29b-2
    3537 mmu-miR-30a 3790 mmu-mir-30a
    3538 mmu-miR-30b 3930 mmu-mir-30b
    3539 mmu-miR-30c 3931 mmu-mir-30c-1
    3932 mmu--mir-30c-2
    3540 mmu-miR-30d 3933 mmu-mir-30d
    3541 mmu-miR-30e 3791 mmu-mir-30e
  • [Table 3-36]
    sequence number miRNA name sequence number miRNA precursor name
    3542 mo-miR-30a 3792 mo-mir-30a
    3543 mo-miR-30b-5p 3934 mo-mir-30b
    3544 mo-miR-30c 3935 mo-mir-30c-1
    3936 mo-mir-30c-2
    3545 mo-miR-30d 1919 mo-mir-30d
    3546 mo-miR-30e 1920 mo-mir-30e
    3547 age-miR-30b 3937 age-mir-30b
    3548 lla-miR-30b 3938 lla-mir-30b
    3549 lla-miR-30c 3939 lla-mir-30c
    3550 mml-miR-30a-5p 1921 mml-mir-30a
    3551 mml-miR-30b 3940 mml-mir-30b
    3552 mml-miR-30c 3941 mml-mir-30c-1
    3942 mml-mir-30c-2
    3553 mml-miR-30d 3943 mml-mir-30d
    3554 mml-miR-30e 3944 mml-mir-30e
    3555 mne-miR-30b 3945 mne-mir-30b
    3556 mne-miR-30c 3946 mne-mir-30c
    3557 mne-miR-30d 3947 mne-mir-30d
    3558 ggo-miR-30a-5p 1922 ggo-mir-30a
    3559 ggo-miR-30b 3948 go-mir-30b
    3560 ggo-miR-30d 3949 mo-mir-30d
    3561 ppa-miR-30a-5p 1923 ppa-mir-30a
    3562 ppa-miR-30b 3950 ppe-mir-30b
    3563 ppa-miR-30d 3951 ppa-mir-30d
    3564 ptr-miR-30a-5p 1924 ptr-mir-30a
    3565 ptr-miR-30b 3952 ptr-mir-30b
    3566 ptr-miR-30c 3953 ptr-mir-30c
    3567 ptr-miR-30d 3954 ptr-mir-30d
    3568 ppy-miR-30a-5p 1925 ppy-mir-30a
    3569 cfa-miR-30a 3955 cfa-mir-30a
    3570 cfa-miR-30b 3956 cfa-mir-30b
    3571 cfa-miR-30c 3957 cfa-mir-30c-1
    3958 cfa-mir-30c-2
    3572 cfa-miR-30d 3959 cfa-mir-30d
    3573 mdo-miR-30a 3960 mdo-mir-30a
    3574 gga-miR-30a-5p 1927 gga-mir-30a
    3575 gga-miR-30b 3961 gga-mir-30b
    3576 gga-miR-30c 3962 gga-mir-30c-1
    3963 gga-mir-30c-2
    3577 gga-miR-30d 3964 gga-mir-30d
    3578 gga-miR-30e 3965 gga-mir-30e
    3579 xtr-miR-30a-5p 1928 xtr-mir-30a
    3580 xtr-miR-30b 3966 xtr-mir-30b
    3581 xtr-miR-30c 3987 xtr-mir-30c-1
    3968 xtr-mir-30c-2
    3582 xtr-miR-30d 3969 xtr-mir-30d
    3583 xtr-miR-30e 3970 xtr-mir-30e
    3584 bta-miR-30a-5p 3971 bta-mir-30a
    3585 bta-miR-30b 3972 bta-mir-30b
    3586 bta-miR-30c 3973 bta-mir-30c
    3587 bta-miR-30d 3974 bta-mir-30d
    3588 bta-miR-30e-5p 3975 bta-mir-30e
    3589 ssc-miR-30c 3976 ssc-mir-30c
    3590 dre-miR-30a 3977 dre-mir-30a
    3591 dre-miR-30b 3978 dre-mir-30b
    3592 de-miR-30c 3979 dre-mir-30c
  • [Table 3-37]
    sequence number miRRA name sequence number miRNA precursor name
    3593 dre-miR-30d 3980 dre-mir-30d
    3594 dre-miR-30e 1929 dre-mir-30e-2
    3595 fru-miR-30b 3981 fru-mir-30b
    3596 fru-miR-30c 3982 fru-mir-30c
    3597 fru-miR-30d 3983 fru-mir-30d
    3598 tni-miR-30b 3984 tni-mir-30b
    3599 tni-miR-30c 3985 tni-mir-30c
    3600 tni-miR-30d 3986 tni-mir-30d
    3601 lla-miR-105 3987 lla-mir-105
    3602 sla-miR-105 3988 sla-mir-105
    3603 mml-miR-105 3989 mml-mir-105-1
    3990 mml-mir-105-2
    3604 mne-miR-105 3991 mne-mir-105
    3605 ggo-miR-105 3992 ggo-mir-105
    3606 ppa-miR-105 3993 ppa-mir-105
    3607 ptr-miR-105 3994 ptr-mir-105
    3608 ppy-miR-105 3995 ppy-mir-105
    3609 ssc-miR-105-1 3996 ssc-mir-105-1
    3997 ssc-mir-105-2
    3610 ssc-miR-105-2 3996 ssc-mir-105-1
    3997 ssc-mir-105-2
    3611 age-miR-124a 3998 age-mir-124a
    3612 lla-miR-124a 3999 lla-mir-124a
    3613 mml-miR-124a 4000 mml-mir-124a-2
    4001 mml-mir-124a-2
    3614 ggo-miR-124a 4002 ggo-mir-124a
    3615 ppa-miR-124a 4003 ppa-mir-124a
    3616 ptr-miR-124a 4004 ptr-mir-124a
    3617 ppy-miR-124a 4005 ppy-mir-124a
    3618 mdo-miR-124a 4006 mdo-mir-124a-1
    4007 mdo-mir-124a-2
    4008 mdo-mir-124a-3
    3619 gga-miR-124a 4009 gga-mir-124a
    3620 gga-miR-124b 4010 gga-mir-124b-1
    4011 gga-mir-124b-2
    3621 xtr-miR-124 4012 xtr-mir-124
    3622 bta-miR-124a 4013 bta-mir-124a
    3623 ssc-miR-124a 4014 ssc-mir-124a
    3624 mmu-miR-150 4015 mmu-mir-150
    3625 mo-miR-150 4016 mo-mir-150
    3626 mml-miR-150 4017 mml-mir-150
    3627 cfa-miR-150 4018 cfa-mir-150
    3628 xtr-miR-150 4019 xtr-mir-150
    3629 bta-miR-150 4020 bta-mir-150
    3630 dre-miR-150 4021 dre-mir-150
    3631 mmu-miR-154 4022 mmu-mir-154
    3632 mo-miR-154 4023 mo-mir-154
    3633 mml-miR-154 4024 mml-mir-154
    3634 mne-miR-154 4025 mne-mir-154
    3635 ggo-miR-154 4026 ggo-mir-154
    3636 ppa-miR-154 4027 ppa-mir-154
    3637 ptr-miR-154 4028 ptr-mir-154
    3638 ppy-miR-154 4029 ppy-mir-154
    3639 mmu-miR-299 3807 mmu-mir-299
    3640 mmi-miR-299-3p 2112 mml-mir-299
    3641 bta-miR-380-5p 4030 bta-mir-380
    3642 mmu-miR-383 4031 mmu-mit-383
    3643 mo-miR-383 4032 mo-mir-383
  • [Table 3-38]
    sequence number miRNA name sequence number miRNA precursor name
    3644 mml-miR-383 4033 mml-mir-383
    3645 cfa-miR-383 4034 cfa-mir-383
    3646 mdo-miR-383 4035 mdo-mir-383
    3647 gga-miR-383 4036 gga-mir-383
    3648 xb-miR-383 4037 xtr-mir-383
    3649 mmu-miR-411 4038 mmu-mir-411
    3650 mo-miR-411 4039 mo-mir-411
    3651 mml-miR-411 4040 mml-mir-411
    3652 mmu-miR-423-3p 4041 mmu-mir-423
    3653 mo-miR-423 4042 mo-mir-423
    3654 mml-miR-423-3p 4043 mml-mir-423
    3655 mmu-miR-433 4044 mmu-mir-433
    3656 mo-miR-433 4045 mo-mir-433
    3657 mml-miR-433 4046 mml-mir-433
    3658 cfa-miR-433 4047 cfa-mir-433
    3659 mml-miR-454 4048 mml-mir-454
    3660 dre-miR-454a 4049 dre-mir-454a
    3661 dre-miR-454b 4050 dre-mir-454b
    3662 mmu-miR-501-5p 4051 mmu-mir-501
    3663 mo-miR-501 4052 mo-mir-501
    3664 mml-miR-501 4053 mml-mir-501
    3665 mmu-miR-504 4054 mmu-mir-504
    3666 mml-miR-504 4055 mml-mir-504
    3667 mml-miR-506 4056 mml-mir-506
    3668 pbi-miR-506 4057 pbi-mir-506
    3669 ptr-miR-506 4058 ptr-mir-506
    3670 ssy-miR-506 4059 ssy-mir-506
    3671 mml-miR-507 4060 mml-mir-507
    3672 pbi-miR-507 4061 pbi-mir-507
    3673 ptr-miR-507 4062 ptr-mir-507
    3674 ssy-miR-507 4063 ssy-mir-507
    3675 ptr-miR-508 4064 ptr-mir-508
    3676 ssy-miR-508 4065 ssy-mir-508
    3677 mml-miR-512-3p 4066 mml-mir-512-1
    4067 mml-mir-512-2
    3678 mml-miR-562 4068 mml-mir-562
    3679 mml-miR-597 4069 mml-mir-597
    3680 mml-miR-605 4070 mml-mir-605
    3681 mml-miR-640 4071 mml-mir-640
    3682 mml-miR-767-5p 4072 mml-mir-767
    3683 mml-miR-770-5p 4073 mml-mir-770
    3684 mml-miR-802 4074 mml-mir-802
    3685 hsa-miR-29c 4075 hsa-mir-29c
    3686 hsa-miR-30c 4076 hsa-mir-30c-1
    4077 hsa-mir-30c-2
    3687 hsa-miR-30d 3092 hsa-mir-30d
    3688 hsa-miR-30e 3093 hsa-mir-30e
    3689 mmu-miR-384-5p 4078 mmu-mir-384
    3690 mo-miR-384-5p 4079 mo-mir-384
    3691 hsa-miR-563 4080 hsa-mir-563
    3692 hsa-miR-130a 3311 hsa-mir-130a
    3693 hsa-miR-130b 4081 hsa-mir-130b
    3694 hsa-miR-301a 4082 hsa-mir-301a
    3695 hsa-miR-301 b 4083 hsa-mir-301b
    3696 mmu-miR-130a 4084 mmu-mir-130a
    3697 mmu-miR-130b 4085 mmu-mir-130b
    3698 mmu-miR-301a 4086 mmu-mir-301a
    3699 mmu-miR-301b 4087 mmu-mir-301b
    3700 mmu-miR-721 4088 mmu-mir-721
  • [Table 3-39]
    sequence number mRNAname sequence number miRNA precursor name
    3701 mo-miR-130a 4089 mo-mir-130a
    3702 mo-miR-130b 4090 mo-mir-130b
    3703 mo-miR-301 a 4091 mo-mir-301a
    3704 mo-miR-301b 4092 mo-mir-301b
    3705 mml-miR-130a 4093 mml-mir-130a
    3706 mml-miR-130b 4094 mml-mir-130b
    3707 mml-miR-301a 4095 mml-mir-301a
    3708 mml-miR-301b 4096 mml-mir-301b
    3709 mme-miR-130a 4097 mne-mir-130a
    3710 ggo-miR-130a 4098 ggo-mir-130a
    3711 ppa-miR-130a 4099 ppa-mir-130a
    3712 cfa-miR-130a 4100 cfa-mir-130a
    3713 cfa-miR-130b 4101 cfa-mir-130b
    3714 mdo-miR-130a 4102 mdo-mir-130a
    3715 gga-miR-130a 4103 gga-mir-130a
    3716 gga-miR-130b 4104 gga-mir-130b
    3717 gga-miR-301 4105 gga-mir-301
    3718 xtr-miR-130a 4106 xtr-mir-130a
    3719 xtr-miR-130b 4107 xtr-mir-130b
    3720 xtr-miR-130c 4108 xtr-mir-130c
    3721 xtr-miR-301 4109 xtr-mir-301-1
    4110 xtr-mir-301-2
    3722 dre-miR-130a 4111 dre-mir-130a-1
    4112 dre-mir-130a-2
    3723 dre-miR-130b 4113 dre-mir-130b
    3724 dre-miR-130c 4114 dre-mir-130c-1
    4115 dre-mir-130c-2
    3725 dre-miR-301a 4116 dre-mir-301a-1
    4117 dre-mir-301a-2
    3726 dre-miR-301b 4118 dre-mir-301b
    3727 dre-miR-301c 4119 dre-mir-301c
    3728 fru-miR-130 4120 fru-mir-130
    3729 fru-miR-301 4121 fru-mir-301
    3730 tni-miR-130 4122 tni-mir-130
    3731 tni-miR-301 4123 tni-mir-301
    3732 mmu-miR-124 4124 mmu-mir-124-1
    4125 mmu-mir-124-2
    4126 mmu-mir-124-3
    3733 mo-miR-124 4127 mo-mir-124-1
    4128 mo-mir-124-2
    4129 mo-mir-124-3
    3734 cfa-miR-124 4130 cfa-mir-124-1
    4131 cfa-mir-124-2
    4132 cfa-mir-124-5
    3735 dre-miR-124 4133 dre-mir-124-1
    4134 dre-mir-124-2
    4135 dre-mir-124-3
    4136 dre-mir-124-4
    4137 dre-mir-124-5
    4138 dre-mir-124-6
    3736 fru-miR-124 4139 fru-mir-124-1
    4140 fru-mir-124-2
    4141 fru-mir-124-3
    3737 tni-miR-124 4142 tri-mir-124-1
    4143 tni-mir-124-2
    4144 tni-mir-124-3
    3738 hsa-miR-557 4145 hsa-mir-557
    3739 mmu-miR-1186 4146 mmu-mir-1186
    3740 xtr-miR-93b 4147 xtr-mir-93b
    3741 hsa-miR-518a-5p 3359 hsa-mir-518a-1
    3360 hsa-mir-518a-2
  • In the present invention, a nucleic acid having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1 to 4147 means a nucleic acid having an identity of at least 90% or more, preferably 93% or more, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, most preferably 98% or more, to a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 3371, as calculated using an analytical software program such as BLAST (J. Mol. Biol., 215, 403 (1990)) or FASTA (Methods in Enzymology, 183, 63 (1990)).
  • As mentioned above, stringent conditions are conditions that allow signals to be detected by adding to a membrane blotted with one strand the other strand labeled with 32P-ATP in a hybridization buffer consisting of 7.5 mL, 0.6 mL of 1M Na2HPO4 (pH 7.2), 21 mL of 10% SDS, 0.6 mL of 50xDenhardt's solution, and 0.3 mL of 10 mg/mL sonicated salmon sperm DNA, carrying out a reaction at 50°C overnight, thereafter washing the membrane with 5xSSC/5% SDS liquid at 50°C for 10 minutes, and further washing the same with 1xSSC/1% SDS liquid at 50°C for 10 minutes, thereafter taking out the membrane, and applying it to an X-ray film.
  • The method of detecting the expression of nucleic acid such as a micro-RNA using a nucleic acid may be any method, as far as the presence of nucleic acid such as a micro-RNA or micro-RNA precursor in a sample can be detected; for example, (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like can be mentioned.
  • The method of detecting the amount expressed of a nucleic acid such as a micro-RNA, a mutation of the nucleic acid, or a mutation of the genome that encodes the nucleic acid using a nucleic acid used in the present invention may be any method, as far as it enables detection of a mutation of the nucleotide sequence of a nucleic acid such as a micro-RNA or micro-RNA precursor in a sample; for example, a method comprising detecting a hetero-double-strand formed by hybridization of a nucleic acid having a non-mutated nucleotide sequence and a nucleic acid having a mutated nucleotide sequence, a method comprising directly sequencing a sample-derived nucleotide sequence to detect the presence or absence of a mutation, and the like can be mentioned.
  • The vector that expresses a nucleic acid used in the present invention may be any vector, as far as it has been designed to biosynthesize a nucleic acid, such as a micro-RNA, used in the present invention, when introduced to, and transcribed in, a cell. As vectors capable of expressing a nucleic acid, such as a micro-RNA, used in the present invention in cells, specifically, pcDNA6.2-GW/miR (manufactured by Invitrogen Company), pSilencer 4.1-CMV (manufactured by Ambion Company), pSINsi-hH1 DNA (manufactured by Takara Bio Company), pSINsi-hU6 DNA (manufactured by Takara Bio Company), pENTR/U6 (manufactured by Invitrogen Company) and the like can be mentioned.
  • The method of suppressing the expression of a gene having a target nucleotide sequence for a nucleic acid, such as a micro-RNA, used in the present invention (hereinafter also referred to as a target gene) may be any method, as far as it suppresses the expression of a gene having the target nucleotide sequence. Here, to suppress the expression encompasses a case where the translation of an mRNA is suppressed, and a case where cleavage or degradation of an mRNA results in a decreased amount of protein translated from the mRNA. As substances that suppress the expression of an mRNA having the target nucleotide sequence, specifically nucleic acids such as siRNAs and antisense oligonucleotides can be mentioned. The siRNAs can be prepared on the basis of information on the continuous sequence of the mRNA (Genes Dev., 13, 3191 (1999)). The number of nucleotide residues constituting one strand of the siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.
  • Micro-RNAs used in the present invention also include an artificial micro-RNA that is a single-stranded RNA 17 to 28 nucleotides long, comprising a sequence complementary to a 7-nucleotide continuous sequence present in a gene (target gene) having a target nucleotide sequence for a micro-RNA of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741 as the 2nd to 8th nucleotides. In case of an RNA comprising an artificial micro-RNA sequence extended anteriorly and posteriorly to form a hairpin structure, wherein the micro-RNA sequence can be cut out from either one strand of the hairpin structure by the biosynthetic pathway of the micro-RNA in the cell, the extended sequence is called an artificial micro-RNA precursor. With the gene whose expression is to be suppressed as a target gene, it is possible to design artificial micro-RNAs and artificial micro-RNA precursors using the method described above.
  • A target nucleotide sequence for a micro-RNA refers to the nucleotide sequence of a nucleic acid consisting of several nucleotides recognized by a micro-RNA used in the present invention, wherein the expression of the mRNA having the nucleotide sequence is suppressed by the micro-RNA. Because an mRNA having a nucleotide sequence complementary to the 2nd to 8th nucleotides from the 5'-end of a micro-RNA undergoes suppression of the translation thereof by the micro-RNA (Current Biology, 15, R458-R460 (2005)), a nucleotide sequence complementary to the 2nd to 8th nucleotides from the 5-end of a micro-RNA used in the present invention can be mentioned as a target nucleotide sequence of the micro-RNA. For example, by providing a target sequence complementary to the 2nd to 8th nucleotides on the 5' terminal side of a micro-RNA, and selecting an mRNA comprising a sequence completely identical to a set of 3' UTR nucleotide sequences of human mRNAs by a method such as character string search, the target nucleotide sequence can be determined. A set of 3' UTR nucleotide sequences of human mRNAs can be prepared using information on genomic sequences and gene positions that can be acquired from "UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway)".
  • As specific examples of genes having a target nucleotide sequence of a micro-RNA of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741, the genes shown in Table 4-1 to Tables 4 to 148, represented by names (Official Symbols and Gene IDs) used in the EntreGene database (http://www.ncbi.nlm.nih.gov/Entrez/) of the US National Center for Biotechnology Information (NCBI), can be mentioned.
  • Figure imgb0001
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  • Figure imgb0006
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  • The substance that controls the expression or a function of a nucleic acid, such as a micro-RNA, used in the present invention, may be any substance such as a low-molecular compound, antibody, or siRNA, as far as it inhibits the expression or a function of the nucleic acid, and an siRNA is particularly preferable. As substances that suppress the expression of a nucleic acid, such as a micro-RNA, used in the present invention, substances that inhibit the function of a factor that is essential to the biosynthesis of the micro-RNA can also be used. Factors that are essential to the biosynthesis of a micro-RNA include, for example, Dicer, TRBP, Exportin5, Drosha, DGCR8 and the like.
  • As a method of expressing a nucleic acid such as a micro-RNA in cells in the present invention, a method using, for example, besides a gene encoding mRNA, a nucleic acid that causes the expression of the micro-RNA and the like when introduced into the cells can be mentioned. As the nucleic acid, a DNA, an RNA, or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used. Specifically, the nucleic acid can be designed in the same way as with Pre-miR miRNA Precursor Molecules (manufactured by Ambion) or miRIDIAN microRNA Mimics (manufactured by GE Healthcare), and the nucleic acid such as the micro-RNA of the present invention can be expressed in cells. Any method can be used to express a micro-RNA, as far as it allows the micro-RNA to be finally formed in the cells; for example, (1) a method wherein a single-stranded RNA that is a precursor of the micro-RNA is introduced, as well as (2) a method wherein an RNA consisting of double-strand consisting of the micro-RNA as it is and a complementary strand for the micro-RNA, which completely complement each other, is introduced, and (3) a method wherein a double-stranded RNA assumed to have resulted from cleavage of a micro-RNA with a Dicer is introduced, can be mentioned. As examples of commercial products based on these methods, miCENTURY OX Precursor (manufactured by B-Bridge), miCENTURY OX siMature (manufactured by B-Bridge), and miCENTURY OX miNatural (manufactured by B-Bridge), respectively, can be mentioned.
  • The method of producing a nucleic acid used in the present invention is not particularly limited; the same can be produced by a method using a publicly known chemical synthesis, or an enzymatic transcription method and the like. As methods using a publicly known chemical synthesis, the phosphoroamidite method, the phosphorothioate method, the phosphotriester method and the like can be mentioned; for example, the same can be synthesized using the ABI3900 high throughput nucleic acid synthesizer (manufactured by Applied Biosystems). As an enzymatic transcription method, transcription with a plasmid or DNA having a desired nucleotide sequence as the template using a typical phage RNA polymerase, for example, T7, T3, or SP6RNA polymerase, can be mentioned.
  • As an example of a method of screening a substance that suppresses the expression or a function of a nucleic acid by using the nucleic acid used in the present invention, a method can be mentioned wherein a vector that expresses the nucleic acid is introduced into cells, and a substance that promotes or suppresses the expression or a function of an RNA having a target nucleotide sequence therefor is screened for.
    A pharmaceutical with a nucleic acid used in the present invention as an active ingredient can be used to diagnose or treat a disease caused by an abnormality of mast cell.
  • As diseases caused by a mast cell abnormality, specifically, atopic dermatitis, asthma, chronic obstructive lung disease, and allergic disease and the like can be mentioned.
    Because abnormalities of mast cells include abnormalities in degranulation, a nucleic acid used in the present invention can be used as a mast cell degranulation control agent, that is, a degranulation promoter or a degranulation suppressant.
  • Mast cell degranulation suppressants are suitably used as prophylactic agents or therapeutic agents for atopic dermatitis, asthma, chronic obstructive pulmonary disease, allergic diseases and the like.
    Mast cell degranulation promoters are suitably used as immunopotentiators.
    The present invention is hereinafter described in detail.
  • 1. Method of detecting the expression of nucleic acids such as micro-RNAs and micro-RNA precursors expressed in mast cells (1-1) Acquisition and cultivation of mast cells
  • The method of acquiring human mast cells is not particularly limited, as far as the human mast cells are acquired safely and efficiently; human mast cells can, for example, be prepared from a human lung, skin, fetal liver and the like by a commonly known method (J. Immunol. Methods, 169, 153 (1994); J. Immunol., 138, 861 (1987); J. Allergy Clin. Immunol., 107, 322 (2001); J. Immunol. Methods., 240, 101 (2000)). Human mast cells can also be prepared by culturing mononuclear cells prepared from human umbilical blood, peripheral blood, bone marrow, lung or skin in the presence of a stem cell factor (hereinafter abbreviated SCF) according to a commonly known method (J. Immunol., 157, 343, (1996); Blood, 91, 187 (1998); J. Allergy Clin. Immunol., 106, 141 (2000); Blood, 97, 1016 (2001); Blood, 98, 1127 (2001); Blood, 100, 3861 (2002); Blood, 97, 2045 (2001)) to allow the mononuclear cells to differentiate into mast cells.
  • A cell line established from a human mast cell can also be used. As human mast cell lines, LAD2 (Leuk. Res., 27, 671 (2003); Leuk. Res., 27, 677 (2003)), which is known to well retain the properties of human mast cells, and the like can be mentioned.
  • (1-2) Acquisition of RNA
  • The method of extracting total RNA from the mast cells acquired by the various methods described above is not particularly limited, as far as a low-molecular RNA such as a micro-RNA is contained; for example, this extraction can be performed by a method described in Molecular Cloning, 3rd edition. Alternatively, the total RNA can also be extracted using Trizol (manufactured by Invitrogen Company), ISOGEN (manufactured by Nippon Gene Company), mirVana miRNA Isolation Kit (manufactured by Ambion Company) , miRNeasy Mini Kit (manufactured by QIAGEN Company) and the like.
  • A low-molecular RNA can also be cloned from a total RNA containing the low-molecular RNA. As a method of cloning a low-molecular RNA, specifically, a method wherein separation and cutting out of a low-molecular RNA by 15% polyacrylamide gel electrophoresis as described in Genes & Development 15, 188-200 (2000), can be mentioned. Then, 5' terminal dephosphorylation, 3'-adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, concatemerization, and ligation to a vector are performed sequentially, thereafter the low-molecular RNA is cloned, and the nucleotide sequence of the clone is determined. Alternatively, for example, a method wherein 5'-adenylation 3'-adapter ligation, 5'-adapter ligation, reverse transcription, PCR amplification, concatemerization, and ligation to a vector are performed sequentially, thereafter the low-molecular RNA is cloned, and the nucleotide sequence of the clone is also determined, as described in Science 294, 858-862 (2001).
  • Alternatively, separation and cleavage of low-molecular RNA by 15% polyacrylamide gel electrophoresis, 5' terminal dephosphorylation, 3'-adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, and ligation to a microbead vector are performed sequentially, thereafter the low-molecular RNA is cloned, and the nucleotide sequence of the microbeads is read to determine the nucleotide sequence, whereby nucleotide sequence information of the low-molecular RNA can also be acquired, as described in Nucleic Acids Research 34, 1765-1771
  • As another method of acquiring a low-molecular RNA, a method involving the use of the low-molecular RNA Cloning Kit (manufactured by Takara Bio Company) can be mentioned.
  • (1-3) Identification of micro-RNA
  • Whether or not the low-molecular RNA sequence is a micro-RNA can be determined on the basis of whether or not the criteria described in RNA, 9, 277-279 (2003) are met. For example, in cases where the low-molecular RNA was newly acquired and the nucleotide sequence thereof was determined, this can be performed as described below.
  • A surrounding genome sequence wherein a DNA sequence corresponding to the nucleotide sequence of the low-molecular RNA acquired is extended by about 50 nucleotides toward the 5' terminal side and the 3' terminal side, respectively, is acquired, and the secondary structure of the RNA expected to be transcribed from the genome sequence is predicted. If the result shows that a hairpin structure is present and the nucleotide sequence of the low-molecular RNA is located in one chain of the hairpin, the low-molecular RNA can be judged to be a micro-RNA. Genome sequences are open to the general public, and are available from, for example, UCSC Genome Bioinformatics (http://genome.ucsc.edu/). For prediction of secondary structures, various programs are open; for example, RNAfold [Nucleic Acids Research 31, 3429-3431 (2003)], Mfold [Nucleic Acids Research 31, 3406-3415 (2003)] and the like can be used. Existing micro-RNA sequences are registered in a database called miRBase (http://microrna.sanger.ac.uk/); whether or not a micro-RNA is identical to an existing micro-RNA can be determined on the basis of whether or not the sequence thereof is identical to one of the sequences listed therein.
  • (1-4) Method of detecting the expression levels of nucleic acid such as micro-RNA
  • As examples of methods of detecting the expression levels of nucleic acid such as micro-RNA, a precursor thereof and the like, methods by (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like can be mentioned.
  • The Northern hybridization is a method wherein a sample-derived RNA is separated by gel electrophoresis, then transferred to a solid support such as a nylon filter, and an appropriately-labeled probe is prepared on the basis of the nucleotide sequence of the nucleic acid of the present invention, and hybridization and washing are performed, whereby a band specifically bound to the nucleic acid is detected; specifically, for example, this method can be performed as described in Science 294, 853-858 (2001) and the like.
  • A labeled probe can be prepared by incorporating a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group and the like in a DNA, RNA, or LNA having a sequence complementary to the nucleotide sequence of the nucleic acid to be used by a method, for example, nick translation, random priming or 5'-terminal phosphorylation. Because the amount of labeled probe bound reflects the expression level of the nucleic acid, the expression level of the nucleic acid can be quantified by quantifying the amount of labeled probe bound. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be achieved by a method described in Molecular Cloning, 3rd edition.
  • Dot blot hybridization is a method wherein an RNA extracted from a tissue or a cell is spotted in dot forms and immobilized on a membrane, and hybridized with a labeled polynucleotide to be a probe, and an RNA that specifically hybridizes with the probe is detected. The probe used may be the same as that used for Northern hybridization. RNA preparation, RNA spotting, hybridization, and RNA detection can be achieved by a method described in Molecular Cloning, 3rd edition.
  • In situ hybridization is a method wherein a paraffin-embedded or cryostat-treated section of a tissue acquired from a living organism, or a cell fixed, is used as a sample and subjected to steps for hybridization with a labeled probe and washing, and the distribution and localization of a nucleic acid in the tissue or cell are examined by microscopic examination [Methods in Enzymology, 254, 419 (1995)]. The probe used may be the same as that used for Northern hybridization. Specifically, a nucleic acid such as micro-RNA etc. can be detected in accordance with a method described in Nature Method 3, 27 (2006).
  • In quantitative PCR, a cDNA synthesized from a sample-derived RNA using a primer for reverse transcription and a reverse transcriptase (hereunder, this cDNA is also referred to as a sample-derived cDNA) is used for the measurement. As a primer for reverse transcription to be supplied for cDNA synthesis, a random primer or a specific RT primer and the like can be used. A specific RT primer refers to a primer having a sequence complementary to a nucleotide sequence corresponding to a nucleic acid and a genomic sequence therearound.
  • For example, a sample-derived cDNA is synthesized, after which a PCR is performed with this cDNA as the template, using a template-specific primer designed from a nucleotide sequence corresponding to a nucleic acid such as micro-RNA or micro-RNA precursor and a genomic sequence therearound, or from a nucleotide sequence corresponding to a primer for reverse transcription, to amplify a cDNA fragment containing the nucleic acid used in the present invention, and the amount of the nucleic acid of the present invention contained in the sample-derived RNA is detected from the number of cycles for reach to a given amount of the fragment. As the template-specific primer, an appropriate region corresponding to a nucleic acid and a genomic sequence therearound is selected, and a pair of a DNA or LNA consisting of a sequence of 20 to 40 nucleotides at the 5' terminus of the nucleotide sequence of the region, and a DNA or LNA consisting of a sequence complementary to a sequence of 20 to 40 nucleotides at the 3' terminus, can be used. Specifically, this can be performed in accordance with a method described in Nucleic Acids Research, 32, e43 (2004) and the like.
  • Alternatively, as the primer for reverse transcription to be supplied for cDNA synthesis, a specific RT primer having a stem-loop structure can also be used. Specifically, this can be performed using a method described in Nucleic Acid Research, 33, e179 (2005), or TaqMan MicroRNA Assays (manufactured by Applied Biosystems).
    Furthermore, by hybridizing a sample-derived cDNA to a DNA, or LNA corresponding to a nucleotide sequence comprising at least one or more of nucleic acids such as micro-RNA, micro-RNA precursor and the like to be used in the present invention immobilized on a substrate such as a filter, glass slide, or silicone, and performing washing, a change in the amount of the nucleic acid can be detected.
    As such methods based on hybridization, methods using differential hybridization [Trends Genet., 7, 314 (1991)] or a microarray (Genome Res., 6, 639 (1996)) can be mentioned. Both methods enable accurate detection of a difference in the amount of a nucleic acid between a control sample and a target sample by immobilizing an internal control, such as a nucleotide sequence corresponding to U6RNA, on a filter or a substrate. Also, by synthesizing labeled cDNAs using differently labeled dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) on the basis of RNAs derived from a control sample and a target sample, and simultaneously hybridizing the two labeled cDNAs to a single filter or a single substrate, accurate quantitation of a nucleic acid can be performed. For example, nucleic acids such as micro-RNA and the like can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004), Nucleic Acid Research, 32, e188 (2004), RNA, 13, 151-159 (2007) and the like. Specifically, a micro-RNA can be detected or quantified in the same manner as mirVana miRNA Bioarray (manufactured by Ambion).
  • In ribonuclease protection assay, first, a promoter sequence such as the T7 promoter or the SP6 promoter is bound to the 3' terminus of a nucleotide sequence corresponding to the nucleic acid such as micro-RNA or micro-RNA precursor to be used in the present invention or a genomic sequence therearound, and a labeled antisense RNA is synthesized with an in vitro transcription system using a labeled NTP (a mixture of ATP, GTP, CTP, and UTP) and an RNA polymerase. The labeled antisense RNA is bound to a sample-derived RNA to form an RNA-RNA hybrid, after which the hybrid is digested with ribonuclease A, which degrades single-stranded RNAs only. The digest is subjected to gel electrophoresis to detect or quantify an RNA fragment protected against the digestion by forming the RNA-RNA hybrid, as a nucleic acid. Specifically, the fragment can be detected or quantified using the mirVana miRNA Detection Kit (manufactured by Ambion).
  • 2. Synthesis of nucleic acid
  • The nucleic acid to be used in the present invention such as a micro-RNA or micro-RNA precursor can synthesize not only an RNA, which is a polymer of a ribonucleotide, but also a DNA, which is a polymer of a deoxyribonucleotide, on the basis of the nucleotide sequences. For example, on the basis of the nucleotide sequence of the micro-RNA identified in 1 above, the nucleotide sequence of a DNA can be determined. The nucleotide sequence of a DNA corresponding to the nucleotide sequence of an RNA can be determined, without exception, by reading the U (uracil) contained in the sequence of the RNA as T (thymine). A polymer being a mixture of a ribonucleotide and a deoxyribonucleotide and a polymer comprising a nucleotide analogue and a derivative of a nucleic acid can also be synthesized in the same manner.
  • The method of synthesizing a nucleic acid used in the present invention is not particularly limited; a method using a publicly known chemical synthesis, or an enzymatic transcription method and the like can be mentioned. As methods using a publicly known chemical synthesis, the phosphoroamidite method, the phosphorothioate method, the phosphotriester method and the like can be mentioned; for example, a nucleic acid can be synthesized using the ABI3900 high throughput nucleic acid synthesizer (manufactured by Applied Biosystems). As an enzymatic transcription method, a method by transcription with a plasmid or DNA having a desired nucleotide sequence as the template using a typical phage RNA polymerase, for example, T7, T3, or SP6RNA polymerase, can be mentioned.
  • 3. Method of detecting a function of a nucleic acid such as micro-RNA or micro-RNA precursor
  • As a method of detecting a function of a nucleic acid such as a micro-RNA, a method can be mentioned wherein the function is determined on the basis of whether or not the translation of the RNA having a target nucleotide sequence is suppressed.
  • Micro-RNAs are known to suppress the translation of an RNA comprising a target nucleotide sequence thereof on the 3' side (3'UTR) [Current Biology, 15, R458-R460 (2005)]. Hence, a DNA wherein a target nucleotide sequence for the single-stranded DNA to be measured is inserted into the 3'UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector, and the expression of the reporter gene is measured when the cell is allowed to express the single-stranded RNA, whereby whether or not a function of a micro-RNA is possessed can be determined.
  • The reporter gene expression vector may be any one, as far as it has a promoter upstream of a reporter gene, and is capable of expressing the reporter gene in the host cell. Any reporter gene can be used; for example, the firefly luciferase gene, the Renilla luciferase gene, the chloramphenicol acetyltransferase gene, the β-glucuronidase gene, the β-galactosidase gene, the β-lactamase gene, the aequorin gene, the green fluorescent protein gene, the DsRed fluorescence gene and the like can be utilized. As examples of reporter gene expression vectors having these properties, psiCHECK-1 (manufactured by Promega), psiCHECK-2 (manufactured by Promega), pGL3-Control (manufactured by Promega), pGL4 (manufactured by Promega), pRNAi-GL (manufactured by Takara Bio Inc.), pCMV-DsRed-Express (manufactured by CLONTECH) and the like can be mentioned. A single-stranded RNA can be expressed by the method described in 5 below.
  • As the function of a micro-RNA of a single-stranded RNA, detection can be achieved as described below. First, host cells are cultured on a multi-well plate and the like, and a reporter gene expression vector having a target sequence and a single-stranded RNA are expressed. Thereafter, reporter activity is measured both when the single-stranded RNA is and is not expressed, based on which the function of micro-RNA can be detected. 4. Method of detecting a mutation of a nucleic acid such as micro-RNA or micro-RNA precursor
    As a method of detecting a mutation of a nucleic acid such as a micro-RNA or micro-RNA precursor, a method can be used wherein a heteroduplex formed by hybridization of a normal type nucleic acid and a mutated type nucleic acid are detected.
  • As methods of detecting a heteroduplex, (1) detection of a heteroduplex by polyacrylamide gel electrophoresis (Trends genet., 7, 5 (1991)), (2) single-strand conformation polymorphism analysis (Genomics, 16, 325-332 (1993)), (3) chemical cleavage of mismatches (CCM) (Human Genetics (1996), Tom Strachan and Andrew P. Read, BIOS Scientific Publishers Limited), (4) enzymatic cleavage of mismatches (Nature Genetics, 9, 103-104 (1996)), (5) denatured gel electrophoresis (Mutat. Res., 288, 103-112 (1993)) and the like can be mentioned.
  • Detection of a heteroduplex by polyacrylamide gel electrophoresis is, for example, performed as described below. First, with a sample-derived DNA or a sample-derived cDNA as a template,
    and using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid used in the present invention, a fragment smaller than 200 nucleotide pairs is amplified. Heteroduplexs, if formed, are slower in mobility than mutation-free homoduplexs, and can be detected as extra bands. In the case of search for a fragment smaller than 200 nucleotide pairs, almost any insertions, deletions, and substitutions of one or more nucleotides can be detected. It is desirable that heteroduplex analysis be performed using a single gel in combination with the single strand conformation analysis described below.
  • In single strand conformation polymorphism analysis (SSCP analysis), a DNA amplified as a fragment smaller than 200 bp with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid of the present invention, is denatured, after which it is electrophoresed in non-denatured polyacrylamide gel. By labeling the primer with an isotope or a fluorescent dye at the time of DNA amplification, or by silver-staining the non-labeled amplification product, the amplified DNA can be detected as a band. To clarify a difference from the wild type pattern, a control sample may be electrophoresed simultaneously, whereby a fragment with a mutation can be detected on the basis of a difference in mobility.
  • In chemical cleavage of mismatches (CCM method), a DNA fragment amplified with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid of the present invention is hybridized to a labeled nucleic acid prepared by allowing a nucleic acid to incorporate an isotope or a fluorescent target, and treated with osmium tetraoxide to cleave one strand of the DNA at the mismatched portion, whereby a mutation can be detected. CCM is one of the most sensitive methods of detection, and can be applied to samples of kilobase length.
  • In place of osmium tetraoxide used above, T4 phage resolvase and an enzyme involved in mismatch repair in cells, such as endonuclease VII, and RNaseA may be used in combination to enzymatically cleave a mismatch. In denaturing gradient gel electrophoresis (DGGE method), a DNA amplified with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid of the present invention is electrophoresed using a gel having a chemical denaturant density gradient or a temperature gradient. The DNA fragment amplified migrates in the gel to a position where it denatures to a single strand, and no longer migrates after the denaturation. Because the migration of the amplified DNA in the gel differs between the presence and absence of a mutation, the presence of the mutation can be detected. To increase the detection sensitivity, the addition of a poly (G:C) end to each primer is effective.
  • By directly determining and analyzing the nucleotide sequence of a sample-derived DNA or sample-derived cDNA, a mutation of a nucleic acid used in the present invention can also be detected.
  • 5. Methods of expressing a nucleic acid such as micro-RNA or micro-RNA precursor
  • A nucleic acid used in the present invention can be expressed by using a vector such that a nucleic acid is transcribed and hence biosynthesized when the vector is introduced into a cell. Specifically, by preparing a DNA fragment comprising a hairpin portion on the basis of the aforementioned nucleotide sequence of a nucleic acid or a genomic nucleotide sequence comprising the foregoing nucleotide sequence, and inserting the fragment downstream of a promoter in the expression vector to construct an expression plasmids, and then introducing the expression plasmid into a host cell suitable for the expression vector, whereby the aforementioned nucleic acid can be expressed.
  • As an expression vector, one capable of self-replication in the host cell, and capable of being incorporated in the chromosome, which comprises a promoter at a position enabling the transcription of a gene comprising the nucleotide sequence of a nucleic acid of the present invention is used. The promoter may be any one, as far as it is capable of expressing in the host cell; for example, a RNA, polymerase II (pol II) system promoter, a RNA polymerase III (pol III) system promoter being a U6RNA and H1RNA transcription system and the like can be mentioned. As examples of pol II system promoters, the promoter of the cytomegalovirus (human CMV) IE (immediate early) gene, the early promoter of SV40 and the like can be mentioned. As examples of expression vectors using them, pCDNA6.2-GW/miR (manufactured by Invitrogen) , pSilencer 4.1-CMV (manufactured by Ambion) and the like can be mentioned. As pol III system promoters, U6RNA, H1RNA or tRNA gene promoters can be mentioned. As examples of expression vectors using them, pSINsi-hH1 DNA (manufactured by Takara Bio Inc.), pSINsi-hU6 DNA (manufactured by Takara Bio Inc.), pENTR/U6 (manufactured by Invitrogen) and the like can be mentioned.
  • Alternatively, by inserting a gene comprising the nucleotide sequence of a nucleic acid used in the present invention into the downstream of the promoter in a viral vector to construct a recombinant viral vector, and introducing the vector into a packaging cell to produce a recombinant virus, the gene comprising the nucleic acid can be expressed.
    The packaging cell may be any cell, as far as it is capable of supplementing a recombinant viral vector deficient in any one of the genes that encode the proteins necessary for the packaging of the virus with the lacked protein; for example, human kidney-derived HEK293 cells, mouse fibroblasts NIH3T3 and the like can be used. As the protein supplemented by the packaging cell, in the case of a retrovirus vector, proteins derived from a mouse retrovirus, such as gag, pol, and env, can be used; in the case of a lentivirus vector, proteins derived from a HIV virus, such as gag, pol, env, vpr, vpu, vif, tat, rev, and nef, can be used; in the case of an adenovirus vector, proteins derived from an adenovirus, such as E1A and E1B, can be used; in the case of an adeno-associated viral vector, proteins such as Rep (p5, p19, p40) and Vp(Cap), can be used.
  • Aside from using an expression vector, it is also possible to introduce a nucleic acid used in the present invention directly to the cell without using a vector. Such nucleic acids useful in this method include not only DNA, RNA or nucleotide analogues, but also chimera molecules thereof, or derivatives of the nucleic acids. Specifically, in the same manner as with Pre-miR mRNA Precursor Molecules (manufactured by Ambion Company) and miRIDIAN microRNA Mimics (manufactured by GE Healthcare Company), a nucleic acid used in the present invention can be expressed. When a micro-RNA is expressed, any method can be used, as far as it allows the micro-RNA to be eventually produced in the cell; examples include methods wherein (1) a single-stranded RNA which is a micro-RNA precursor, (2) an RNA, consisting of a double strand consisting of a micro-RNA and a strand complementary to the micro-RNA which are completely complementary to each other, (3) or a double-stranded RNA assuming a state after the micro-RNA is cut by a Dicer, is introduced. Available products involving the use of these methods are miCENTURY OX Precursor (manufactured by B-Bridge Company), miCENTURY OX siMature (manufactured by B-Bridge Company), and miCENTURY OX miNatural (manufactured by B-Bridge Company).
  • 6. Methods of suppressing the activity of a nucleic acid such as micro-RNA or micro-RNA precursor
  • The activation of a nucleic acid such as micro-RNA or micro-RNA precursor to be used in the present invention can be suppressed using an antisense technology (Bioscience and Industry, 50, 322 (1992); Kagaku, 46, 681 (1991), Biotechnology, 9, 358 (1992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992); Cell Technology, 16, 1463 (1997)], triple helix technology (Trends in Biotechnology, 10, 132 (1992)), ribozyme technology (Current Opinion in Chemical Biology, 3, 274 (1999), FEMS Microbiology Reviews, 23, 257 (1999), Frontiers in Bioscience, 4, D497 (1999), Chemistry & Biology, 6, R33 (1999), Nucleic Acids Research, 26, 5237 (1998), Trends In Biotechnology, 16, 438 (1998)), decoy DNA method (Nippon Rinsho - Japanese Journal of Clinical Medicine, 56, 563 (1998), Circulation Research, 82, 1023 (1998), Experimental Nephrology, 5, 429 (1997), Nippon Rinsho - Japanese Journal of Clinical Medicine, 54, 2583 (1996)), or a siRNA (short interfering RNA).
  • An antisense refers to one that allows nucleotide sequence-specific hybridization of a nucleic acid having a nucleotide sequence complementary to a certain target nucleic acid to enable the suppression of the expression of the target nucleic acid. As the nucleic acid used as the antisense, a DNA, an RNA or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used. Specifically, by preparing an antisense in accordance with a method described in Nature 432, 226 (2004) and the like, the expression can be suppressed. In addition, in the same way as with Anti-miR miRNA Inhibitors (manufactured by Ambion) and miRIDIAN miRNA Inhibitors (manufactured by GE Healthcare), the expression of a nucleic acid used in the present invention can be suppressed.
  • An siRNA refers to a short double-stranded RNA comprising the nucleotide sequence of a certain target nucleic acid, that is capable of suppressing the expression of the target nucleic acid by RNA interference (RNAi). The sequence of an siRNA can be designed as appropriate from the target nucleotide sequence on the basis of conditions shown in the literature (Genes Dev., 13, 3191 (1999)). By synthesizing two RNAs having a 19-nucleotide sequence selected and a complementary sequence, with TT added to the 3' terminus of each thereof using a nucleic acid synthesizer, and performing annealing, an siRNA can be prepared. By inserting a DNA corresponding to the above-described 19-nucleotide sequence selected into an siRNA expression vector such as pSilencer 1.0-U6 (manufactured by Ambion) or pSUPER (manufactured by OligoEngine), a vector that expresses an siRNA capable of suppressing the expression of the gene can be prepared. Although any siRNA can be used to suppress a function of a nucleic acid, such as a micro-RNA, used in the present invention, as far as it is capable of suppressing the function of the nucleic acid, preference is given to an siRNA designed on the basis of sequence information on SEQ ID NO:1 to 3371. The number of nucleotide residues constituting one strand of the siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.
  • Using an antisense or siRNA specific for a nucleic acid such as micro-RNA expressed in mast cells, or a precursor of the micro-RNA, the expression of a micro-RNA expressed in mast cells, a precursor of the micro-RNA and the like can be suppressed. For example, by administering an antisense oligonucleotide or siRNA specific for the micro-RNA or the micro-RNA precursor, the activation of the micro-RNA can be suppressed, and the action of the micro-RNA or micro-RNA precursor in mast cells can be controlled.
  • In addition, in the case of a patient affected by an abnormality of the expression of a micro-RNA expressed in mast cells or a precursor thereof, by administering an antisense oligonucleotide or siRNA specific for the micro-RNA or precursor thereof to the patient, it is possible to control a function of mast cells to treat a disease that develops as a result of the above-described expression abnormality. Hence, an antisense oligonucleotide or siRNA that is specific for the micro-RNA or precursor thereof is useful as a therapeutic agent for a disease caused by a cell abnormality of mast cells.
  • When an antisense oligonucleotide or siRNA specific for a nucleic acid such as a micro-RNA or a precursor thereof is used as the above-described therapeutic agent, the antisense oligonucleotide or siRNA alone, or a nucleic acid that encodes the same after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, or adeno-associated virus vector, may be prepared as a pharmaceutical preparation according to the conventional method described in 9 below, and administered.
  • 7. Methods of suppressing functions or expression of target genes using a nucleic acid such as micro-RNA or micro-RNA precursor
  • Any method can be used to suppress a function or the expression of the target gene for a nucleic acid used in the present invention, as far as it is based on an activity of a nucleic acid such as a micro-RNA to suppress the expression of the mRNA having the target nucleotide sequence. For example, a method can be mentioned wherein a nucleic acid used in the present invention such as a micro-RNA is expressed to increase the amount of the nucleic acid such as the micro-RNA in cells, whereby the translation of the mRNA having the target sequence is suppressed to suppress the expression of the gene. The expression of a nucleic acid used in the present invention can be achieved by the method described in 5 above. As examples of mRNAs having a target nucleotide sequence for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1-1543, and 3372-3741, the respective sets of genes shown in the foregoing Table 4 can be mentioned.
  • Using an siRNA against a target gene shown in Table 4, it is possible to suppress a function of the target gene.
  • 8. Methods of screening for substances that controls the expression or a function of a nucleic acid such as a micro-RNA or micro-RNA precursor using the nucleic acid
  • A substance that controls, that is, promotes or suppresses the expression or a function of a nucleic acid such as a micro-RNA or a precursor thereof can be screened for using a nucleic acid used in the present invention. For example, a nucleotide sequence to be targeted for screening is chosen from the nucleotide sequence of a micro-RNA or a precursor thereof to be used in the present invention and by means of cells that express a nucleic acid having the nucleotide sequence, a substance that promotes or suppresses the expression or a function of the chosen micro-RNA or precursor thereof can be screened for.
  • As cells that express a nucleic acid having the nucleotide sequence of a micro-RNA or micro-RNA precursor, used for screening, mast cells, as well as transformant cells obtained by introducing a vector that expresses a nucleic acid having the nucleotide sequence into a host cell such as an animal cell or yeast, cells incorporating a nucleic acid having the nucleotide sequence introduced directly without using a vector and the like as described in 5 above can also be used.
  • As specific screening methods, a method comprising using a change in the expression level of a nucleic acid such as an micro-RNA or precursor thereof to be targeted in the screening as an index, as well as a method comprising using a change in the expression level of an mRNA having a target sequence for a nucleic acid such as a micro-RNA or a gene product encoded thereby as an index can be mentioned.
  • (a) Screening method comprising using a change in the expression level of a micro-RNA or precursor thereof to be targeted in the screening as an index
  • A test substance is brought into contact with a cell that expresses the nucleic acid, and with a change in the expression level of the nucleic acid selected as an index, a substance that promotes or suppresses the nucleic acid such as a micro-RNA and precursor thereof is obtained. The expression level of a nucleic acid can be detected by the method described in 3 above.
  • (b) Screening method comprising using a change in the expression level of an mRNA having a target sequence for a nucleic acid such as a micro-RNA to be targeted in the screening or a gene product encoded thereby as an index
  • A test substance is brought into contact with a cell that expresses the mRNA, and with a change in the expression level of an mRNA having a target sequence of the nucleic acid selected or a gene product encoded thereby as an index, a substance that promotes or suppresses the expression or a function of a nucleic acid such as a micro-RNA and a precursor thereof can be obtained. Alternatively, a DNA having a target sequence of a nucleic acid of the present invention such as micro-RNA inserted into the 3' UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector, a test substance is brought into contact with the cell, and with a change in the expression level of the reporter gene as an index, a substance that promotes or suppresses the expression or a function of a nucleic acid such as a micro-RNA and a precursor thereof can be obtained.
  • Choice of a target sequence can be achieved by the method described in 7 above; as examples of an mRNA having a target sequence of a nucleic acid such as a micro-RNA consisting of the nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741, the above-described genes shown in Table 4 above can be exemplified.
  • 9. Mast cell degranulation control agents comprising a nucleic acid such as a micro-RNA or a micro-RNA precursor
  • Nucleic acids such as micro-RNAs and micro-RNA precursors and nucleic acids having nucleotide sequences complementary to the nucleotide sequences thereof, used in the present invention, can be used as mast cell degranulation control agents, that is, degranulation suppressants or degranulation promoters, because they control the expression of genes having a target sequence.
  • As active ingredients of mast cell degranulation suppressants, the nucleic acids (a) to (h) below can be mentioned.
    1. (a) A nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 70, 151 to 581, 1238 to 1461, and 3407 to 3452.
    2. (b) A nucleic acid of 17 to 28 nucleotides comprising a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 70, 151 to 581, 1238 to 1461, and 3407 to 3452.
    3. (c) A nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1 to 70, 151 to 581, 1238 to 1461, and 3407 to 3452.
    4. (d) A nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 70, 151 to 581, 1238 to 1461, and 3407 to 3452.
    5. (e) A nucleic acid comprising the 2nd to 8th nucleotides of a nucleotide sequence of any one of SEQ ID NOs:1 to 70, 151 to 581, 1238 to 1461, and 3407 to 3452.
    6. (f) A nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 1621, 1634, 1665 to 1668, 1688, 1709 to 2196, 2862 to 2864, 2978 to 3287, 3365, and 3780 to 3828.
    7. (g) A nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1544 to 1621, 1634, 1665 to 1668, 1688, 1709 to 2196, 2862 to 2864, 2978 to 3287, 3365, and 3780 to 3828.
    8. (h) A nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 1621, 1634, 1665 to 1668, 1688, 1709 to 2196, 2862 to 2864, 2978 to 3287, 3365, and 3780 to 3828.
    As the above-described nucleic acids, micro-RNAs and micro-RNA precursors are suitably used.
  • Substances that promote a function or the expression of the nucleic acids (a) to (h) above can also be used as mast cell degranulation suppressants.
    Substances that suppress a function or the expression of a target gene for the nucleic acids (a) to (h) above can also be used as mast cell degranulation suppressants. As substances that suppress the expression of a target gene, an siRNA against the mRNA of the target gene, an antisense against the target gene, and the like can be mentioned.
  • Meanwhile, substances that suppress a function or the expression of the nucleic acids (i) to (p) below can also be used as mast cell degranulation suppressants. As substances that suppress a function or the expression of the nucleic acids, siRNAs and antisenses against the nucleic acids, and the like can be mentioned.
    As active ingredients of mast cell degranulation promoters, the nucleic acids (i) to (p) below can be mentioned.
    • (i) A nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:71 to 150, 582 to 1237, 1462 to 1543, 3372 to 3406, and 3453 to 3741.
    • (j) A nucleic acid of 17 to 28 nucleotides comprising a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:71 to 150, 582 to 1237, 1462 to 1543, 3372 to 3406, and 3453 to 3741.
    • (k) A nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:71 to 150, 582 to 1237, 1462 to 1543, 3372 to 3406, and 3453 to 3741.
    • (l) A nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:71 to 150, 582 to 1237, 1462 to 1543, 3372 to 3406, and 3453 to 3741.
    • (m) A nucleic acid comprising the 2nd to 8th nucleotides of a nucleotide sequence of any one of SEQ ID NOs:71 to 150, 582 to 1237, 1462 to 1543, 3372 to 3406, and 3453 to 3741.
    • (n) A nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1557, 1575, 1590, 1592 to 1593, 1622 to 1708, 1919 to 1925, 1927 to 1929, 2112, 2197 to 2977, 3092 to 3093, 3162 to 3163, 3169, 3177, 3281, 3288 to 3371, 3742 to 3779, 3790 to 3792, 3807, and 3829 to 4147.
    • (o) A nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1557, 1575, 1590, 1592 to 1593, 1622 to 1708, 1919 to 1925, 1927 to 1929, 2112, 2197 to 2977, 3092 to 3093, 3162 to 3163, 3169, 3177, 3281, 3288 to 3371, 3742 to 3779, 3790 to 3792, 3807, and 3829 to 4147.
    • (p) A nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1557, 1575, 1590, 1592 to 1593, 1622 to 1708, 1919 to 1925, 1927 to 1929, 2112, 2197 to 2977, 3092 to 3093, 3162 to 3163, 3169, 3177, 3281, 3288 to 3371, 3742 to 3779, 3790 to 3792, 3807, and 3829 to 4147.
    As the above-described nucleic acids, micro-RNAs and micro-RNA precursors are suitably used.
  • Substances that promote a function or the expression of the nucleic acids (i) to (p) above can also be used as mast cell degranulation promoters.
    Substances that suppress a function or the expression of a target gene for the nucleic acids (i) to (p) above can also be used as mast cell degranulation promoters. As substances that suppress the expression of a target gene, an siRNA against the mRNA of the target gene, an antisense against the target gene and the like can be mentioned.
  • Meanwhile, substances that suppress a function or the expression of the nucleic acids (a) to (h) above can also be used as mast cell degranulation promoters. As substances that suppress a function or the expression of the nucleic acids, an siRNA and antisense against the nucleic acids and the like can be mentioned.
    As a mast cell degranulation control agent of the present invention, a vector that expresses one of the nucleic acids (a) to (h) and (i) to (p) above can also be used.
  • Regarding the preparation forms, methods of administration and the like for the mast cell degranulation control agents of the present invention, the same applies as with the diagnostic agents and therapeutic agents containing a nucleic acid such as a micro-RNA, or a micro-RNA precursor, described in 10 below.
  • 10. Diagnostic agents and therapeutic agents containing a nucleic acid such as a micro-RNA or a micro-RNA precursor
  • Nucleic acids, such as micro-RNAs and micro-RNA precursors, used in the present invention can be used as therapeutic agents for diseases resulting from a mast cell abnormality and the like, because they control the expression of genes having a target sequence, or control the expression of nucleic acids, such as micro-RNAs, used in the present invention. Also, siRNAs against a target gene for the nucleic acids can be used as therapeutic agents for diseases resulting from a mast cell abnormality and the like because they control the expression of the gene. As mast cell abnormalities, abnormalities of mast cell differentiation and degranulation, inflammatory mediator production, cytokine production, chemokine production and the like can be mentioned; as diseases caused thereby, atopic dermatitis, asthma, chronic obstructive lung disease, allergic disease and the like can be mentioned.
  • By quantifying a nucleic acid of the present invention or detecting a mutation therein, a disease resulting from a mast cell abnormality and the like can be diagnosed.
    A diagnostic agent comprising a nucleic acid of the present invention, according to the desired diagnostic method, may comprise reagents necessary for quantitation or detection of mutation of_a nucleic acid used in the present invention, for example, buffering agents, salts, reaction enzymes, labeled proteins that bind to_a nucleic acid of the present invention, and a color developer for detection and the like.
  • Although a therapeutic agent containing as an active ingredient a nucleic acid used in the present invention can be administered alone, the same is normally desirably administered as a pharmaceutical preparation produced by an optionally chosen method known well in the technical field of pharmaceutical making with one or more pharmacologically acceptable carriers blended therein.
    The route of administration used is desirably the most effective one in treatment; oral administration, or parenteral administration such as intraoral administration, airway administration, intrarectal administration, subcutaneous administration, intramuscular administration and intravenous administration can be mentioned, and desirably intravenous administration can be mentioned.
  • As dosage forms, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injection formulations, ointments, tapes and the like can be mentioned.
    As preparations appropriate for oral administration, emulsions, syrups, capsules, tablets, powders, granules and the like can be mentioned.
    Liquid preparations like emulsions and syrups can be produced using water, saccharides such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil, antiseptics such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor and peppermint and the like as additives.
  • Capsules, tablets, powders, granules and the like can be produced using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropylcellulose, and gelatin, surfactants such as fatty acid esters, plasticizers such as glycerin and the like as additives.
  • As appropriate preparations for parenteral administration, injection formulations, suppositories, sprays and the like can be mentioned.
    An injection formulation is prepared using a carrier consisting of a salt solution, a glucose solution or a mixture of both and the like. A suppository is prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. A spray is prepared using a carrier that does not stimulate the recipient's oral cavity and airway mucosa, and that disperses the active ingredients as fine particles to facilitate the absorption thereof, and the like.
  • As examples of the carrier, specifically, lactose, glycerin and the like can be exemplified. Depending on the nature of the nucleic acid used in the present invention, and of the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, components exemplified as additives for oral preparations can also be added.
    The dose or frequency of administration varies depending on desired therapeutic effect, method of administration, duration of treatment, age, body weight and the like, and is normally 10 µg/kg to 20 mg/kg per day for an adult.
  • A therapeutic agent containing as an active ingredient a nucleic acid used in the present invention can also be produced by blending a vector that expresses the nucleic acid used in the present invention and a base for nucleic acid-based therapeutic agents (Nature Genet., 8, 42(1994)).
    The base used in the therapeutic agent of the present invention may be any base for ordinary use in injection formulations; distilled water, solutions of salts such as sodium chloride or a mixture of sodium chloride and an inorganic salt, solutions of mannitol, lactose, dextran, glucose and the like, solutions of amino acids such as glycine and arginine, mixed solutions of organic acid solutions or salt solutions and glucose solution and the like can be mentioned. In accordance with a conventional method, using auxiliary agents such as an osmoregulator, a pH adjuster, a vegetable oil such as sesame oil or soybean oil, lecithin, and a surfactant such as a non-ionic surfactant in these bases, an injection formulation may be prepared as a solution, suspension, or dispersion. These injection formulations can also be prepared as preparations for dissolution before use, by procedures such as powdering and lyophilization. A therapeutic agent of the present invention can be used for treatment as is in the case of a liquid, or after being dissolved in a base described above, optionally sterilized, in the case of a solid, just before treatment.
  • As a vector that expresses a nucleic acid used in the present invention, the recombinant viral vector prepared by the method described in 5 above can be mentioned, more specifically, a retrovirus vector and a lentivirus vector and the like can be mentioned.
    In addition, by combining a nucleic acid used in the present invention with a polylysine-conjugated antibody that is specific for adenovirus hexon protein to prepare a complex, and binding the complex obtained to an adenovirus vector, a viral vector can be prepared. The viral vector is capable of stably reaching the desired cell, being incorporated into cells by endosome, being decomposed in the cells, and efficiently expressing the nucleic acid.
  • A viral vector based on Sendai virus, which is a (-) strand RNA virus, has been developed ( WO97/16538 , WO97/16539 ); using the Sendai virus, a Sendai virus vector that expresses a nucleic acid used in the present invention can be prepared.
    A nucleic acid used in the present invention can also be introduced by a non-viral nucleic acid introduction method. The same can be introduced by, for example, calcium phosphate coprecipitation (Virology, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)], microinjection method (Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92-94 (1981)), membrane fusion-mediated introduction mediated by liposome (Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989); Hum. Gene Ther., 3, 267-275, (1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-2011 (1992)) or direct DNA uptake and receptor-mediated DNA introduction method (Science, 247, 1465-1468 (1990); J. Biol. Chem., 266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659 (1991); J. Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990); Proc. Natl. Acad. Sci. USA, 88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA, 87, 4033-4037 (1990); Proc. Natl. Acad. Sci. USA, 88, 8850-8854 (1991); Hum. Gene Ther., 3, 147-154 (1991)) and the like.
  • Membrane fusion-mediated introduction mediated by liposome allows a nucleic acid used in the present invention to be incorporated locally in the tissue, and to be expressed, by administering a liposome preparation directly to the target tissue (Hum. Gene Ther., 3, 399 (1992)). For direct targeting of a nucleic acid to a focus, a technology including direct uptake of nucleic acid is preferable.
    For receptor-mediated nucleic acid introduction, for example, a method performed by binding a nucleic acid to a protein ligand via polylysine can be mentioned. A ligand is chosen on the basis of the presence of a corresponding ligand receptor on the cell surface of the desired cell or tissue. The ligand-nucleic acid conjugate can be injected directly into a blood vessel as desired, and can be directed to a target tissue wherein receptor binding and nucleic acid-protein complex internalization occur. To prevent the destruction of the nucleic acid in a cell, an adenovirus may be infected simultaneously to destroy the endosome function.
  • 11. Method of measuring the degree of mast cell activation
  • The fact that a nucleic acid such as a micro-RNA or a micro-RNA precursor exhibits at least one of the actions of activation suppression, degranulation suppression, inflammatory mediator production suppression, cytokine production suppression, and chemokine production suppression on mast cells can be confirmed by, for example, introducing a nucleic acid used in the present invention or an siRNA against a target gene for the nucleic acid, into a mast cell, thereafter stimulating the mast cell, measuring a released substance such as (i) histamine or p-hexosaminidase, which can be an index of degranulation, (ii) an inflammatory mediator such as LTC4, LTD4, LTE4, or PGD2, (iii) a cytokine such as TNF-α or GM-CSF, or (iv) a chemokine such as IL-8, I-209, or MIP-1α, and comparing the result with that obtained when the nucleic acid used in the present invention, or the siRNA against the target gene for the nucleic acid, is not introduced.
  • Also, by introducing a nucleic acid, micro RNA or micro-RNA precursor used in the present invention, or an siRNA for a target gene of the micro-RNA, into a mast cell, and detecting the induction of apoptosis by a measurement of the fragmentation of chromatin DNA, the TUNEL method and the like, the fact that the siRNA possesses apoptosis inducing action can be confirmed.
    The fact can also be confirmed by introducing a nucleic acid used in the present invention or an siRNA against a target gene for the nucleic acid into a mast cell in the presence of a substance that suppresses the activation of mast cells, thereafter stimulating the mast cell, and comparing the result with that obtained when the nucleic acid used in the present invention or the siRNA against a target gene for the nucleic acid is not introduced.
  • As example of methods of stimulating a mast cell, a method wherein an anti-IgE antibody is added after the cell is cultured with the addition of IgE, a method wherein Compound 48/80 is added, a method wherein polymyxin B is added, a method wherein dextran is added, a method wherein a calcium ionophore is added, a method wherein acetylcholine is added, a method wherein carbachol is added, a method wherein thrombin is added, a method wherein concanavalin A is added, a method wherein a calcium ionophore is added, a method wherein ATP is added, a method wherein doxorubicin is added, and the like can be mentioned. As examples of substances that suppress the activation of mast cells, substances that inhibit the process wherein a micro-RNA is biosynthesized from a micro-RNA precursor can be mentioned.
  • Mast cell activation can also be examined by measuring, in place of degranulation, production of cytokines such as TNF-α and GM-CSF, production of chemokines such as IL-8, 1-309, and MIP-1α, production of inflammatory mediators such as LTC4, LTD4, LTE4, and PGD2 and the like [Blood, 100, 3861(2002)].
    The present invention is hereinafter described specifically by means of the following Examples. However, the present invention is not limited to these Examples.
  • [Example 1] Action of forcible expression of micro-RNA precursor on degranulation of human mast cells
  • A human micro-RNA precursor was introduced into the human mast cell line LAD2, and the influence of the micro-RNA precursor on the degranulation was examined.
    LAD2, a recently established human mast cell line, is known to well retain the nature of human mast cells (Leuk. Res., 27, 671 (2003); Leuk. Res., 27, 677 (2003)). LAD2 was obtained from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD 20892-1881, USA), and cultured using Stem Pro-34 medium (manufactured by Invitrogen Company) containing 100 ng/mLSCF.
  • LAD2 was seeded to a 96-well plate at 4×103 cells per well, a micro-RNA precursor was introduced using a lipofection method, specifically TransIT-TKO (manufactured by Mirus Company), to obtain a final concentration of 25 nM. The micro-RNA precursors used were human micro-RNA library Ver.1 and Ver.2 synthesized at Ambion Company. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • Two days after the introduction of the micro-RNA precursor by the lipofection method, human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 µg/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO2 concentration. The following day, a rabbit antihuman IgE antibody (manufactured by DAKO Company) was added to obtain a final concentration of 10 µg/mL, and the cells were incubated for 30 minutes in an incubator at 37°C and a 5% CO2 concentration to induce degranulation. Degrees of degranulation were evaluated by measuring the β-hexosaminidase activity of the enzyme in the granules released in the medium. A measurement of the β-hexosaminidase activity was performed by adding to the medium 40 µL of 20 mmol/L 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (manufactured by Sigma Company) dissolved in the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl2, 0.6 mmol/L MgCl2, 0.6 mmol/L KH2PO4, 10 mM HERPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), incubating the cells at 37°C for 3 hours, thereafter determining the absorbance at 450 nm using the plate reader EnVision (manufactured by Perkin-Elmer Company). After the measurement, Triton X-100 was added to the medium to obtain a final concentration of 1%, and the same experiment was performed, whereby the total β-hexosaminidase activity contained in LAD2 was measured. The ratio of degranulation induced by the anti-IgE antibody was calculated as the ratio (%) to the total β-hexosaminidase activity released with 1% Triton X-100. The relative activity of degranulation was evaluated by the deviation from the median by a calculation of subtracting the median from the degranulation efficiency, and dividing the difference by the mean deviation. Degranulation promoting activity was expressed with plus symbols, and degranulation suppressing activity with minus symbols.
    Two independent experiments were performed; the results are shown in Table 5-1 to Table 5-2.
  • [Table 5-1]
    introduced micro-RNA relative activity of degranulalion
    first time second time
    hsa-miR-593 -4.42 -4.65
    hsa-miR-34a -3.51 -3.04
    hsa-miR-634 -3.47 -3.29
    hsa-miR-449b -3.17 -2.23
    hsa-miR-654 -3.03 -0.73
    hsa-miR-140 -2.92 -0.81
    hsa-miR-25 -2.78 -3.02
    hsa-miR-338 -2.70 -0.86
    hsa-miR-199a -2.61 -2.78
    hsa-miR-658 -2.59 -1.49
    hsa-miR-328 -2.57 -1.18
    hsa-miR-587 -2.57 -1.62
    hsa-miR-208 -2.51 -0.61
    hsa-miR-214 -2.46 -1.51
    hsa-miR-199b -2.40 -1.95
    hsa-miR-18a -2.40 -3.94
    hsa-miR-660 -2.36 -1.60
    hsa-miR-595 -2.33 -1.55
    hsa-miR-525* -2.32 -1.67
    hsa-miR-647 -2.11 -1.29
    hsa-miR-625 -2,10 -2.65
    hsa-miR-520d -2.09 -0.10
    hsa-miR-18b -2.06 -3.26
    hsa-miR-21 -2.06 -1.07
    hsa-miR-210 -2.02 -0.48
    hsa-miR-345 -1.91 -1.70
    hsa-miR-765 -1.88 -1.85
    hsa-miR-329 -1.85 -1.53
    hsa-miR-218 -1.77 -2.81
    hsa-miR-604 -1.76 -1.53
    hsa-miR-635 -1.75 -2.26
    hsa-miR-668 -1.62 -1.78
    hsa-miR-573 -1.58 -2.16
    hsa-miR-517* -1.41 -2.38
    hsa-miR-637 -1.13 -2.38
    hsa-miR-211 -1.10 -1.39
    hsa-miR-17-3p -0.76 -2.05
    hsa-miR-515-3p -0.74 -2.34
    hsa-miR-486 -0.69 -2.72
    hsa-miR-16 -0.65 -3.31
    hsa-miR-485-5p -0.14 -2.48
    hsa-miR-510 -0.09 -2.68
    hsa-miR-200a* 0.31 2.32
    hsa-miR-378 0.48 4.12
    hsa-miR-223 0.66 2.50
    hsa-miR-596 0.66 2.05
    hsa-miR-409-3p 0.68 2.65
    hsa-miR-27b 0.70 2.85
    hsa-miR-449 0.70 -2.11
    hsa-miR-613 0.79 2.12
    hsa-miR-146a 0.93 2.09
    hsa-miR-429 0.97 2.08
    hsa-miR-544 1.09 2.43
  • [Table 5-2]
    introduced micro-RNA relative activity of degranulation
    first time second time
    hsa-miR-617 1.12 2.57
    hsa-miR-106a 1.17 2.07
    hsa-miR-632 1.26 2.74
    hsa-miR-374 1.57 1.55
    hsa-miR-135b 1.72 1.56
    hsa-miR-181a 1.73 1.91
    hsa-miR-296 1.77 1.52
    hsa-miR-550 1.80 2.37
    hsa-miR-133b 1.85 2.06
    hsa-miR-376a 1.94 2.80
    hsa-miR-206 2.06 3.09
    hsa-miR-28 2.08 2.01
    hsa-miR-127 2.09 1.28
    hsa-miR-518c 2.09 0.68
    hsa-miR-450 2.11 0.08
    hsa-miR-487b 2.12 2.67
    hsa-miR-362 2.12 -0.08
    hsa-let-7e 2.45 0.46
    hsa-miR-33b 2.47 2.39
    hsa-miR-216 2.62 1.84
    hsa-miR-373* 2.80 0.32
    hsa-miR-182* 2.82 1.18
    hsa-miR-146b 2.87 1.63
    hsa-miR-1 3.23 3.69
    hsa-miR-659 3.71 3.41
    hsa-miR-126* 3.73 4.52
  • Seven days after the introduction of the micro-RNA precursor as well, human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 µg/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO2 concentration; the following day, degranulation was induced by the addition of a rabbit antihuman IgE antibody, and the ratio of degranulation was measured. The results are shown in Table 6-1 to Table 6-2.
  • [Table 6-1]
    introduced micro-RNA relative activity of degranulation
    first time second time
    hsa-miR-593 -6.23 -4.03
    hsa-miR-634 -3.91 -3.29
    hsa-miR-647 -3.14 -1.75
    hsa-miR-595 -2.70 -1.60
    hsa-miR-190 -2.50 -2.22
    hsa-miR-587 -2.48 -1.87
    hsa-miR-658 -2.43 -2.46
    hsa-miR-25 -2.33 -3.04
    hsa-miR-329 -2.32 -1.69
    hsa-miR-520h -2.25 -0.68
    hsa-miR-195 -2.23 -1.44
    hsa-miR-325 -2.22 -1.55
    hsa-miR-449b -2.19 -1.66
    hsa-miR-199a -2.16 -3.53
    hsa-miR-425-5p -2.15 -1.82
    hsa-miR-299-5p -2.07 -1.53
    hsa-miR-202* -2.05 -1.08
    hsa-miR-211 -2.04 -1.47
    hsa-miR-520f -1.96 -2.00
    hsa-miR-26b -1.94 -2.07
    hsa-miR-32 -1.93 -1.92
    hsa-miR-484 -1.72 -2.47
    hsa-miR-612 -1.64 -2.45
    hsa-miR-214 -1.59 -1.69
    hsa-miR-30a-3p -1.59 -1.98
    hsa-miR-522 -1.58 -1.53
    hsa-miR-515-5p -1.50 -2.18
    hsa-miR-199b -1.49 -2.49
    hsa-miR-107 -1.45 -2.09
    hsa-miR-24 -1.33 -2.63
    hsa-miR-20b -1.26 -2.12
    hsa-miR-488 -1.18 -2.37
    hsa-miR-675 -1.13 -2.71
    hsa-miR-766 -0.86 -4.41
    hsa-miR-486 -0.57 -2.18
    hsa-miR-148b -0.15 -2.08
    hsa-miR-514 -0.04 2.18
    hsa-miR-650 0.14 -2.34
    hsa-miR-524* 0.64 2.92
    hsa-miR-133a 0.74 2.55
    hsa-miR-432* 0.82 2.09
    hsa-miR-448 0.85 3.29
    hsa-miR-489 0.93 2.03
    hsa-miR-192 1.02 2.37
    hsa-miR-550 1.18 2.54
    hsa-miR-128b 1.31 2.78
    hsa-miR-31 1.42 2.10
    hsa-miR-542-3p 1.51 2.68
    hsa-miR-27b 1.52 2.58
    hsa-miR-22D 1.53 1.82
    hsa-miR-409-3p 1.53 2.36
    hsa-miR-33b 1.54 2.07
    hsa-miR-187 1.54 1.71
  • [Table 6-2]
    introduced micro-RNA relative activity of degranulation
    first time second time
    hsa-miR-452 1.69 2.11
    hsa-miR-450 1.72 1.79
    hsa-miR-517c 1.79 2.62
    hsa-miR-451 1.85 2.15
    hsa-miR-609 1.88 1.81
    hsa-miR-215 1.92 3.26
    hsa-miR-552 1.92 1.64
    hsa-miR-100 1.94 1.75
    hsa-miR-409-5p 2.04 0.84
    hsa-miR-608 2.04 0.37
    hsa-miR-142-5p 2.07 2.50
    hsa-miR-224 2.09 2.21
    hsa-miR-601 2.10 1.63
    hsa-miR-545 2.11 1.97
    hsa-miR-28 2.12 1.26
    hsa-miR-96 2.16 1.90
    hsa-miR-129 2.16 3.08
    hsa-miR-487b 2.20 2.61
    hsa-let-7e 2.25 -0.14
    hsa-miR-223 2.26 1.23
    hsa-miR-181a 2.26 1.38
    hsa-miR-801 2.27 1.90
    hsa-miR-136 2.31 0.22
    hsa-miR-183 2.34 1.66
    hsa-miR-9 2.35 1.94
    hsa-miR-302c* 2.37 0.92
    hsa-miR-133b 2.38 3.02
    hsa-miR-1 2.39 2.59
    hsa-miR-769-5p 2.40 1.18
    hsa-miR-627 2.46 1.55
    hsa-miR-302b* 2.47 -0.25
    hsa-miR-222 2.54 2.14
    hsa-miR-217 2.65 0.86
    hsa-miR-378 2.66 2.93
    hsa-miR-23b 2.71 -1.04
    hsa-miR-644 2.88 1.45
    hsa-miR-182* 3.11 4.32
    hsa-miR-216 3.34 0.70
    hsa-miR-659 3.77 2.95
  • These results revealed that the ratio of degranulation decreased with the introduction of micro-RNA precursors corresponding to hsa-miR-16, 195, 17-3p, 18a, 18b, 20b, 21, 24, 25, 32, 26b, 30a-3p, 34a, 449, 449b, 107, 140, 148b, 190, 199a, 199b, 202*, 208, 210, 211, 214, 218, 299-5p, 325, 328, 329, 338, 345, 425-5p, 484, 485-5p, 486, 488, 510, 515-3p, 515-5p, 517*, 520d, 520f, 520h, 522, 525*, 573, 587, 593, 595, 604, 612, 625, 634, 635, 637, 647, 650, 654, 658, 660, 668, 675, 765, and 766 (these are nucleic acids consisting of the nucleotide sequences of SEQ ID NOs:1 to 66, respectively), and conversely the ratio of degranulation increased with the introduction of micro-RNA precursors corresponding to hsa-let-7e, hsa-miR-1, 206, 613, 9, 23b, 27b, 28, 31, 33b, 96, 100, 106a, 126*, 127, 128b, 129, 133a, 133b, 135b, 136, 142-5p, 146a, 181a, 182*, 183, 187, 192, 215, 200a*, 216, 217, 220, 222, 223, 224, 296, 302b*, 302c*, 362, 373*, 374, 376a, 378, 409-3p, 409-5p, 429, 432*, 448, 450, 451, 452, 487b, 489, 514, 517c, 518c, 524*, 542-3p, 544, 545, 550, 552, 596, 601, 608, 609, 617, 627, 632, 644, 659, 769-5p, and 801 (these are nucleic acids consisting of the nucleotide sequences of SEQ ID NOs:71 to 118 and 120 to 146).
  • A measurement of degranulation activity using a 6-well plate was also performed. LAD2 was seeded to a 6-well plate at 5×105 cells per well, a micro-RNA precursor was introduced using a lipofection method, specifically Gene Silencer (manufactured by Genlantis Company), to obtain a final concentration of 25 nM. The micro-RNA, precursors used were Pre-miR™miRNA Precursor Molecules synthesized at Ambion Company. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • Two days after the introduction of the micro-RNA precursor by the lipofection method, 1.0 µg/mL human myeloma IgE (manufactured by Cosmo Bio Company) was added, and the cells were cultured overnight in an incubator at 37°C and a 5% CO2 concentration. The following day, the medium was removed via centrifugation, and the plate was washed with the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl2, 0.6 mmol/L MgCl2, 0.6 mmol/L KH2PO4, 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), after which the cells were suspended by the addition of 1.5 mL of the Tyrode buffer solution, and the suspension was dispensed to a 96-well plate at 100 µL per well. Subsequently, a rabbit antihuman IgE antibody (manufactured by DAKO Company) was added to obtain a final concentration of 10 µg/mL, and the cells were incubated for 20 minutes in an incubator at 37°C and a 5% CO2 concentration to induce degranulation. The supernatant was recovered via centrifugation, and the β-hexosaminidase activity in the supernatant was measured to determine the degree of degranulation.
  • The β-hexosaminidase activity was evaluated by adding to 50 µL of the recovered supernatant 50 µL of 4 mmol/L p-nitrophenyl-N-acetyl-β-glucosaminide (manufactured by Sigma Company) dissolved in 40 mmol/L citrate solution (pH 4.5), and incubating the supernatant at 37°C for 1 hour, thereafter measuring the absorbance of a sample supplemented with 100 µL of 0.2 mol/L glycine (pH 10.7) at 405 nm using the plate reader 1420 ARVOsx (manufactured by Perkin-Elmer Company). Separately, Triton X-100 was added at a final concentration of 1% in place of the rabbit antihuman IgE antibody, and the same experiment was performed, whereby the total β-hexosaminidase activity in LAD2 was measured. The ratio of degranulation was calculated as the ratio (%) of the β-hexosaminidase activity in the supernatant to the total β-hexosaminidase activity, and the relative activity of degranulation was calculated for each precursor with the ratio of degranulation in a negative control plot (Gene Silencer only) taken as 1.0.
  • Seven days after the introduction of the micro-RNA precursor, human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 1.0 µg/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO2 concentration; the following day, a rabbit antihuman IgE antibody was added to induce degranulation, the ratio of degranulation was measured, and the relative activity of degranulation was calculated for each precursor with the ratio of degranulation in a negative control plot (Gene Silencer only) taken as 1.0.
    The results for the relative activity of degranulation at 3 days and 8 days after the introduction of each micro-RNA precursor are shown in Table 7.
  • [Table 7]
    introduced mism-RNA/siRNA relative activity of degranulation
    3 days later 5 days later
    hsa-miR-197 0.52 0.62
    hsa-miR-221 0.60 0.63
    hsa-miR-200a 1.17 2.16
    hsa-miR-200c 1.64 2.73
    hsa-miR-142-3p 1.71 2.94
    hsa-miR-361 1.82 1.13
  • As shown in Table 7, it was found that the ratio of degranulation increased with the introduction of micro-RNA precursors corresponding to hsa-miR-200c, 142-3p, 200a, and 361 (these are nucleic acids consisting of the nucleotide sequences of SEQ ID NOs:119 and 148 to 150, respectively), and that conversely the ratio of degranulation decreased with the introduction of hsa-miR-197 and 221 (these are nucleic acids consisting of the nucleotide sequences of SEQ ID NOs:69 and 70, respectively).
  • [Example 2] Degranulation activity in human mast cells with micro-RNA antisense introduced thereto
  • Each of antisense oligonucleotides against hsa-miR-194, hsa-miR-500 and hsa-miR-365 (these are nucleic acids consisting of the nucleotide sequences of SEQ ID NOs: 67, 68 and 147, respectively) was introduced into LAD2 using a lipofection method to obtain a final concentration of 25 nM. The micro-RNA antisense oligonucleotides used were Anti-miR™miRNA Inhibitors (manufactured by Ambion Company). Lipofection was performed according to the method described in the instruction manual attached to the product.
  • Two days after the introduction of the micro-RNA antisense oligonucleotide by the lipofection method, human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 µg/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO2 concentration. The following day, a rabbit antihuman IgE antibody (manufactured by DAKO Company) was added to obtain a final concentration of of 10 µg/mL, and the cells were incubated for 30 minutes in an incubator at 37°C and a 5% CO2 concentration to induce degranulation. Degrees of degranulation were evaluated by measuring the β-hexosaminidase activity of the enzyme in the granules released into the medium. The measurement of the β-hexosaminidase activity was performed by adding to the medium 40 µL of 20 mmol/L 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (manufactured by Sigma Company) dissolved in the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl, 0.6 mmol/L MgCl2, 0.6 mmol/L KH2PO4, 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), and incubating the cells at 37°C for 3 hours, thereafter determining the absorbance at 450 nm using the plate reader Envision (manufactured by Perkin-Elmer Company).
  • After the measurement, Triton X-100 was added to the medium to obtain a final concentration of 1%, and the same experiment was performed, whereby the total β-hexosaminidase activity contained in LAD2 was measured. The mean for the wells with no cells seeded thereto was calculated, and this was subtracted as the background from each measured value. The ratio of degranulation induced by the anti-IgE antibody was calculated as the ratio (%) of the β-hexosaminidase activity to the total β-hexosaminidase activity released with 1% Triton X-100. The relative activity of degranulation was evaluated by the deviation from the median by a calculation of subtracting the median from the degranulation efficiency, and dividing the difference by the mean deviation. Degranulation promoting activity was expressed with plus symbols, and degranulation suppressing activity with minus symbols. Two independent experiments were performed; the results are shown in Table 8.
  • [Table 8]
    introduced antisense relative active of degranulation,
    first time second time
    hsa-miR-194 3.69 2.67
    hsa-miR-500 4.42 2.36
    hsa-miR-365 -2.35 -2.16
  • Seven days after the introduction of the antisense oligonucleotide against the micro-RNA, human myeloma IgE (manufactured by Cosmo Bio Company) was added to obtain a final concentration of 0.3 µg/mL, and the cells were cultured overnight in an incubator at 37°C and a 5% CO2 concentration; the following day, a rabbit antihuman IgE antibody was added to induce degranulation, and the ratio of degranulation was measured. The results are shown in Table 9.
  • [Table 9]
    introduced antisense relative activity of degranulation
    first time second time
    hsa-miR-194 2.63 2.05
    hsa-miR-500 3.75 2.82
    hsa-miR-365 -0.94 -0.77
  • As shown in Tables 8 and 9, it was found that the ratio of degranulation increased with the introduction of the antisense oligonucleotide against hsa-miR-194 or hsa-miR-500, and that conversely the ratio of degranulation decreased with the introduction of the antisense oligonucleotide against hsa-miR-365.
  • [Example 3] Action of forcible expression of micro-RNA precursor on degranulation in mast cells in degranulation-suppressed state
  • An siRNA of the Dicerl gene (hereinafter, the Dicerl-siRNA) and a human micro-RNA precursor were co-transfected to LAD2, and the influence of the micro-RNA precursor on the degranulation stimulated by Compound 48/80 was examined.
  • LAD2 was seeded to a 96-well plate at 2×104 cells per well, and the Dicerl-siRNA and the micro-RNA precursor were co-transfected using a lipofection method, specifically GeneSilencer (manufactured by Genlantis Company), to obtain a final concentration of 25 nM of each. The sequence of the siRNA against the human Dicerl gene used was the Dicerl-siRNA that targets SEQ ID NO:4148 (manufactured by QIAGEN Company). The micro-RNA precursor used was a human micro-RNA library synthesized at Ambion Company. As negative controls for the siRNA and the micro-RNA precursor, All Stars Negative Control siRNA (hereinafter, siRNA allstars) (manufactured by QIAGEN Company) and Pre-miR™miRNA Precursor Negative Control #1 (hereinafter, miR-negacon#1) (manufactured by Ambion Company), respectively, were provided, and were introduced into LAD2 in the same manner. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • Three days after the introduction of the micro-RNA precursor by the lipofection method, the culture medium was replaced with the Tyrode buffer solution (126.1 mmol/L NaCl, 4.0 mmol/L KCl, 1.0 mmol/L CaCl2, 0.6 mmol/L MgCl2, 0.6 mmol/L KH2PO4, 10 mM HEPES, 5.6 mmol/L D-glucose, 0.1% bovine serum albumin, pH 7.4), and the cells were suspended. Subsequently, equal volumes of the cell suspension were seeded to two different sites in a 96-well plate, and Compound 48/80 (manufactured by Sigma-Aldrich Company) at a final concentration of 1.0 µg/mL and Triton X-100 at a final concentration of 1%, respectively, were added. After incubation at 37°C for 20 minutes to induce degranulation, centrifugation was performed, and the culture supernatant was recovered.
  • Degrees of degranulation were evaluated by measuring the β-hexosaminidase activity of the enzyme in the granules released into the culture supernatant. The β-hexosaminidase activity was evaluated by adding to the recovered culture supernatant 50 mL of 4 mmol/L 4-nitrophenyl-N-acetyl-β-D-glucosaminide (manufactured by Sigma-Aldrich Company) dissolved in 40 mmol/L citric acid buffer solution (pH 4.5), and incubating the supernatant at 37°C for 1 hour, thereafter determining the absorbance of a sample supplemented with 100 mL of 0.2 mol/L glycine (pH 10.7) at 405 nm using the plate reader 1420 ARVOsx (manufactured by Perkin-Elmer Company).
  • The ratio of degranulation induced by Compound 48/80 was calculated as the ratio (%) to the β-hexosaminidase activity in the plot with the addition of 1% Triton X-100. The activity of the micro-RNA for cancelling the degranulation suppression due to introduction of the Dicerl-siRNA was calculated as a relative activity with the ratio of degranulation in the plot with co-transfection of the Dicerl-siRNA and miR-negacon#1 taken as 0%, and with the ratio of degranulation in the plot with co-transfection of the siRNA allstars and micro-RNA-negacon#1 taken as 100%. The results are shown in Table 10.
  • [Table 10]
    introduced micro-RNA relative activity of degranulation
    first time second time
    hsa-miR-29b 153.1 115.1
    hsa-miR-589 152.2 ND
    hsa-miR-299-3p 131.2 94.1
    hsa-miR-29a 120.6 88.1
    hsa-miR-30b 118.2 58.3
    hsa-miR-506 117.3 97.2
    hsa-miR-105 117.3 110.1
    hsa-miR-127 113.8 167.3
    hsa-miR-769-5p 104.7 ND
    hsa-miR-597 104.4 ND
    hsa-miR-504 102.1 63.9
    hsa-miR-770-5p 101.4 ND
    hsa-miR-150 101.2 90.4
    hsa-miR-605 101.0 ND
    hsa-miR-640 100.0 ND
    hsa-miR-154 99.7 151.0
    hsa-miR-380-5p 97.7 13.6
    hsa-miR-629 92.6 ND
    hsa-miR-660 92.4 ND
    hsa-miR-128b 90.0 112.9
    hsa-miR-518c 90.0 -40.0
    hsa-miR-767-5p 88.5 ND
    hsa-miR-423 87.1 11.1
    hsa-miR-567 87.1 ND
    hsa-miR-33a-5p 86.2 60.9
    hsa-miR-383 84.2 -16.9
    hsa-miR-497 82.8 33.3
    hsa-miR-124a 82.3 83.4
    hsa-miR-512-3p 81.8 117.5
    hsa-miR-133a 81.0 63.5
    hsa-miR-501 75.9 65.6
    hsa-miR-133b 74.1 103.8
    hsa-miR-514 69.7 50.5
    hsa-miR-619 64.8 151.1
    hsa-miR-527 62.9 55.0
    hsa-miR-454-3p 61.3 54.9
    hsa-miR-148a 60.5 82.0
    hsa-miR-411 58.4 71.9
    hsa-miR-128a 58.4 96.1
    hsa-miR-511 58.1 71.2
    hsa-miR-508 55.3 91.1
    hsa-miR-562 53.8 81.3
    hsa-miR-802 52.2 58.2
    hsa-miR-146a 52.1 56.1
    hsa-miR-373* 31.5 82.1
    hsa-miR-433 22.6 84.7
    hsa-miR-507 -6.2 107.4
  • [Example 4] mRNA expression analysis of LAD2 forcibly overexpressing miR-142-3p precursor and miR-24 precursor
  • Each of the miR-142-3p and miR-24 precursors of human micro-RNA was introduced into LAD2, and gene clusters the amounts of whose expression fluctuate due to introduction of each of the micro-RNA, precursors were comprehensively searched for using an RNA array.
    LAD2 was seeded to a 6-well plate at 5×105 cells per well, and each micro-RNA precursor was introduced using a lipofection method, specifically GeneSilencer (manufactured by Genlantis Company), to obtain a final concentration of 30 nM. The micro-RNA precursors used were Pre-miR™ miRNA Precursor Molecules synthesized at Ambion Company. For negative control, Pre-miR™RNA Precursor Negative Control#2 manufactured by Ambion Company (hereinafter, miR-negacon#2) was used. Lipofection was performed according to the method described in the instruction manual attached to the product.
  • Two days after the introduction of each of the micro-RNA precursors by the lipofection method, the cells were recovered, and total RNA was purified from the cells using miRNeasy (manufactured by QIAGEN Company).
    The purified total RNA was analyzed for mRNA expression using the Whole Human Genome Oligo Microarray manufactured by Agilent Technologies Company. The analytical procedure was in accordance with the protocol of Agilent Technologies Company. Specifically, a cRNA obtained by labeling the total RNA with Cy3 was hybridized to the microarray at 65°C for about 17 hours, and the microarray was washed, after which the microarray was scanned at a resolution of 5 µm using the Agilent technologies Microarray Scanner to acquire signal values.
  • To examine the gene clusters exhibiting an expressional fluctuation in the plots with the introduction of the miR-142-3p or miR-24 precursor, respectively, the relative activity of the amount expressed was calculated with the signal value for each gene detected in the plot with the introduction of miR-negacon#2 taken as 1.0. In the plots with the introduction of the miR-142-3p precursor or the miR-24 precursor, gene clusters whose expression was suppressed to the extent of a relative activity of 0.8 or less are shown in Table 11 and Table 12-1 to Table 12-3, respectively.
  • Figure imgb0149
  • Figure imgb0150
  • Figure imgb0151
  • Figure imgb0152
  • Industrial Applicability
  • According to the present invention, a mast cell degranulation control agent, a diagnostic agent or therapeutic agent for a disease resulting from mast cell degranulation control, a method of controlling mast cell degranulation, and a screening method for a mast cell degranulation control agent can be provided. These are useful in the diagnosis or treatment of a disease resulting from mast cell degranulation control.
  • [Free text of sequence listing]
    • SEQ ID NO:3916 - n represents a, c, g or u
    • SEQ ID NO:4148 - explanation of artificial sequences: synthetic DNAs

Claims (18)

  1. A mast cell degranulation control agent comprising as an active ingredient any one of the nucleic acids (a) to (h) below:
    (a) a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
    (b) a nucleic acid of 17 to 28 nucleotides comprising a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
    (c) a nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
    (d) a nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1 to 1543 and 3372 to 3741,
    (e) a nucleic acid comprising the 2nd to 8th nucleotides of a nucleotide sequence of any one of SEQ ID Nos:1 to 1543 and 3372 to 3741,
    (f) a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147,
    (g) a nucleic acid consisting of a nucleotide sequence having an identity of 90% or more to a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147, and
    (h) a nucleic acid that hybridizes under stringent conditions with a complementary strand for a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs:1544 to 3371 and 3742 to 4147.
  2. The mast cell degranulation control agent according to claim 1, wherein the nucleic acid is a micro-RNA or a micro-RNA precursor.
  3. A mast cell degranulation control agent comprising as an active ingredient a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid of claim 1.
  4. A mast cell degranulation control agent comprising as an active ingredient a double-stranded nucleic acid consisting of the nucleic acid of claim 1 and a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of the former nucleic acid.
  5. A mast cell degranulation control agent comprising as an active ingredient a vector that expresses the nucleic acid of any one of claims 1 to 3 or the double-stranded nucleic acid of claim 4.
  6. A mast cell degranulation control agent comprising as an active ingredient a substance that controls the expression or a function of the nucleic acid of claim 1.
  7. The mast cell degranulation control agent according to claim 6, wherein the substance that controls the expression or a function of the nucleic acid is an siRNA or an antisense oligonucleotide.
  8. A mast cell degranulation control agent comprising as an active ingredient a substance that suppresses the expression of a target gene for the nucleic acid of claim 1.
  9. The mast cell degranulation control agent according to claim 8, wherein the substance that suppresses the expression of a target sequence for a nucleic acid is an siRNA or an antisense oligonucleotide.
  10. A diagnostic agent or therapeutic agent for a disease resulting from an abnormality of mast cells, comprising as an active ingredient the nucleic acid of any one of claims 1 to 3, the double-stranded nucleic acid of claim 4, the vector of claim 5, or the substance of any one of claims 6 to 9.
  11. A diagnostic agent for a disease resulting from an abnormality of mast cells, comprising as an active ingredient a reagent for detecting the amount expressed of the nucleic acid of claim 1, a mutation of the nucleic acid or a mutation of the genome that encodes the nucleic acid.
  12. The diagnostic agent or therapeutic agent according to claim 10 or 11, wherein the disease resulting from an abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic diseases.
  13. A method for treating a disease resulting from an abnormality of mast cells, comprising administering an effective amount of the degranulation suppressant of any one of claims 1 to 9 to a subject in need thereof.
  14. The method according to claim 13, wherein the disease resulting from an abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic diseases.
  15. A use of the degranulation suppressant of any one of claims 1 to 9 for producing a therapeutic agent for a disease resulting from an abnormality of mast cells.
  16. The use according to claim 15, wherein the disease resulting from an abnormality of mast cells is a disease selected from the group consisting of atopic dermatitis, asthma, chronic obstructive pulmonary disease and allergic diseases.
  17. A method of controlling mast cell degranulation, comprising using the nucleic acid of any one of claims 1 to 3, the double-stranded nucleic acid of claim 4, the vector of claim 5, or the substance of any one of claims 6 to 9.
  18. A screening method for a mast cell degranulation control agent, wherein the promotion or suppression of the expression or a function of the nucleic acid of claim 1 serves as an index.
EP09758404A 2008-06-04 2009-06-04 Nucleic acid capable of controlling degranulation of mast cell Withdrawn EP2298359A1 (en)

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